Monoclonal Antibody

ABSTRACT

The present invention is related to methods and compositions for the therapeutic and diagnostic use in the treatment of diseases and disorders which are caused by or associated with amyloid or amyloid-like proteins including amyloidosis, a group of disorders and abnormalities associated with amyloid protein such as Alzheimer&#39;s disease. The present invention provides novel methods and compositions comprising highly specific and highly effective antibodies having the ability to specifically recognize and bind to specific epitopes from a range of β-amyloid proteins. The antibodies enabled by the teaching of the present invention are particularly useful for the treatment of diseases and disorders which are caused by or associated with amyloid or amyloid-like proteins including amyloidosis, a group of diseases and disorders associated with amyloid plaque formation including secondary amyloidosis and age-related amyloidosis including, but not limited to, neurological disorders such as Alzheimer&#39;s Disease (AD).

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.12/213,006, filed Jun. 12, 2008, now U.S. Pat. No. 8,613,923, whichclaims the benefit of U.S. Provisional Application No. 60/943,541, filedJun. 12, 2007, and U.S. Provisional Application No. 60/943,790, filedJun. 13, 2007, each of which is incorporated by reference herein in itsentirety.

The present invention is related to methods and compositions for thetherapeutic and diagnostic use in the treatment of diseases anddisorders which are caused by or associated with amyloid or amyloid-likeproteins including amyloidosis, a group of disorders and abnormalitiesassociated with amyloid protein such as Alzheimer's disease.

Amyloidosis is not a single disease entity but rather a diverse group ofprogressive disease processes characterized by extracellular tissuedeposits of a waxy, starch-like protein called amyloid, whichaccumulates in one or more organs or body systems. As the amyloiddeposits build up, they begin to interfere with the normal function ofthe organ or body system. There are at least 15 different types ofamyloidosis. The major forms are primary amyloidosis without knownantecedent, secondary amyloidosis following some other condition, andhereditary amyloidosis.

Secondary amyloidosis occurs in people who have a chronic infection orinflammatory disease, such as tuberculosis, a bacterial infection calledfamilial Mediterranean fever, bone infections (osteomyelitis),rheumatoid arthritis, inflammation of the small intestine (granulomatousileitis), Hodgkin's disease, and leprosy.

Amyloid protein fibrils, which account for about 90% of the amyloidmaterial, comprise one of several different types of proteins. Certainof these proteins are capable of folding into so-called “beta-pleated”sheet fibrils, a unique protein configuration which exhibits bindingsites for Congo red resulting in the unique staining properties of theamyloid protein. In addition, amyloid deposits are closely associatedwith the amyloid P (pentagonal) component (AP), a glycoprotein relatedto normal serum amyloid P (SAP), and with sulfated glycosaminoglycans(GAG), complex carbohydrates of connective tissue.

Many diseases of aging are based on or associated with amyloid-likeproteins and are characterized, in part, by the buildup of extracellulardeposits of amyloid or amyloid-like material that contribute to thepathogenesis, as well as the progression of the disease. These diseasesinclude, but are not limited to, neurological disorders such asAlzheimer's Disease (AD), including diseases or conditions characterizedby a loss of cognitive memory capacity such as, for example, mildcognitive impairment (MCI), Lewy body dementia, Down's syndrome,hereditary cerebral hemorrhage with amyloidosis (Dutch type); the GuamParkinson-Dementia complex. Other diseases which are based on orassociated with amyloid-like proteins are progressive supranuclearpalsy, multiple sclerosis; Creutzfeld Jacob disease, Parkinson'sdisease, HIV-related dementia, ALS (amyotropic lateral sclerosis),inclusion-body myositis (IBM), Adult Onset Diabetes; senile cardiacamyloidosis; endocrine tumors, and others, including maculardegeneration.

Although pathogenesis of these diseases may be diverse, theircharacteristic deposits often contain many shared molecularconstituents. To a significant degree, this may be attributable to thelocal activation of pro-inflammatory pathways thereby leading to theconcurrent deposition of activated complement components, acute phasereactants, immune modulators, and other inflammatory mediators (McGeeret al., 1994).

Alzheimer's Disease (AD) is a neurological disorder primarily thought tobe caused by amyloid plaques, an accumulation of abnormal deposit ofproteins in the brain. The most frequent type of amyloid found in thebrain of affected individuals is composed primarily of Aβ fibrils.Scientific evidence demonstrates that an increase in the production andaccumulation of beta-amyloid protein in plaques leads to nerve celldeath, which contributes to the development and progression of AD. Lossof nerve cells in strategic brain areas, in turn, causes reduction inthe neurotransmitters and impairment of memory. The proteins principallyresponsible for the plaque build up include amyloid precursor protein(APP) and two presenilins (presenilin I and presenilin II). Sequentialcleavage of the amyloid precursor protein (APP), which is constitutivelyexpressed and catabolized in most cells, by the enzymes β and γsecretase leads to the release of a 39 to 43 amino acid Aβ peptide. Thedegradation of APPs likely increases their propensity to aggregate inplaques. The Aβ(1-42) fragment in particular has a high propensity ofbuilding aggregates due to two very hydrophobic amino acid residues atits C-terminus. The Aβ(1-42) fragment is therefore believed to be mainlyinvolved and responsible for the initiation of neuritic plaque formationin AD and to have, therefore, a high pathological potential. There istherefore a need for specific antibodies that can target and diffuseamyloid plaque formation.

The symptoms of AD manifest slowly and the first symptom may only bemild forgetfulness. In this stage, individuals may forget recent events,activities, the names of familiar people or things and may not be ableto solve simple math problems. As the disease progresses, symptoms aremore easily noticed and become serious enough to cause people with AD ortheir family members to seek medical help. Mid-stage symptoms of ADinclude forgetting how to do simple tasks such as grooming, and problemsdevelop with speaking, understanding, reading, or writing. Later stageAD patients may become anxious or aggressive, may wander away from homeand ultimately need total care.

Presently, the only definite way to diagnose AD is to identify plaquesand tangles in brain tissue in an autopsy after death of the individual.Therefore, doctors can only make a diagnosis of “possible” or “probable”AD while the person is still alive. Using current methods, physicianscan diagnose AD correctly up to 90 percent of the time using severaltools to diagnose “probable” AD. Physicians ask questions about theperson's general health, past medical problems, and the history of anydifficulties the person has carrying out daily activities. Behavioraltests of memory, problem solving, attention, counting, and languageprovide information on cognitive degeneration and medical tests such astests of blood, urine, or spinal fluid, and brain scans can provide somefurther information.

The management of AD consists of medication-based and non-medicationbased treatments. Treatments aimed at changing the underlying course ofthe disease (delaying or reversing the progression) have so far beenlargely unsuccessful. Medicines that restore the deficit (defect), ormalfunctioning, in the chemical messengers of the nerve cells(neurotransmitters), in particular the cholinesterase inhibitors (ChEIs)such as tacrine and rivastigmine, have been shown to improve symptoms.ChEIs impede the enzymatic degradation of neurotransmitters therebyincreasing the amount of chemical messengers available to transmit thenerve signals in the brain.

For some people in the early and middle stages of the disease, the drugstacrine (COGNEX®, Morris Plains, N.J.), donepezil (ARICEPT®, Tokyo, JP),rivastigmine (EXELON®, East Hanover, N.J.), or galantamine (REMINYL®,New Brunswick, N.J.) may help prevent some symptoms from becoming worsefor a limited time. Another drug, memantine (NAMENDA®, New York, N.Y.),has been approved for treatment of moderate to severe AD. Medicationsare also available to address the psychiatric manifestations of AD.Also, some medicines may help control behavioral symptoms of AD such assleeplessness, agitation, wandering, anxiety, and depression. Treatingthese symptoms often makes patients more comfortable and makes theircare easier for caregivers. Unfortunately, despite significant treatmentadvances showing that this class of agents is consistently better than aplacebo, the disease continues to progress, and the average effect onmental functioning has only been modest. Many of the drugs used in ADmedication such as, for example, ChEIs also have side effects thatinclude gastrointestinal dysfunction, liver toxicity and weight loss.

Other diseases that are based on or associated with the accumulation anddeposit of amyloid-like protein are mild cognitive impairment, Lewy bodydementia (LBD), amyotrophic lateral sclerosis (ALS), inclusion-bodymyositis (IBM) and macular degeneration, in particular age-relatedmacular degeneration (AMD).

Mild cognitive impairment (MCI) is a general term most commonly definedas a subtle but measurable memory disorder. A person with MCIexperiences memory problems greater than normally expected with aging,but does not show other symptoms of dementia, such as impaired judgmentor reasoning. MCI is a condition that frequently reflects a preclinicalstage of AD.

The deposition of β-amyloid within the entorhinal cortex (EC) isbelieved to play a key role in the development of mild cognitiveimpairment (MCI) in the elderly. This is in line with the observationthat the CSF-A Aβ(1-42) levels decline significantly once AD becomesclinically overt. In contrast to CSF-Aβ(1-42) CSF-tau levels aresignificantly increased in the MCI stage, and these values continue tobe elevated thereafter, indicating that increased levels of CSF-tau mayhelp in detecting MCI subjects who are predicted to develop AD.

Lewy body dementia (LBD) is a neurodegenerative disorder that can occurin persons older than 65 years of age, which typically causes symptomsof cognitive (thinking) impairment and abnormal behavioural changes.Symptoms can include cognitive impairment, neurological signs, sleepdisorder, and autonomic failure. Cognitive impairment is the presentingfeature of LBD in most cases. Patients have recurrent episodes ofconfusion that progressively worsen. The fluctuation in cognitiveability is often associated with shifting degrees of attention andalertness. Cognitive impairment and fluctuations of thinking may varyover minutes, hours, or days.

Lewy bodies are formed from phosphorylated and nonphosphorylatedneurofilament proteins; they contain the synaptic proteinalpha-synuclein as well as ubiquitin, which is involved in theelimination of damaged or abnormal proteins. In addition to Lewy Bodies,Lewy neurites, which are inclusion bodies in the cell processes of thenerve cells, may also be present. Amyloid plaques may form in the brainsof patients afflicted with LBD, however they tend to be fewer in numberthan seen in patients with Alzheimer's disease. Neurofibrillary tangles,the other micropathological hallmark of AD, are not a maincharacteristic of LBD but are frequently present in addition to amyloidplaques.

Amyotrophic lateral sclerosis (ALS) is characterized by degeneration ofupper and lower motor neurons. In some ALS patients, dementia or aphasiamay be present (ALS-D). The dementia is most commonly a frontotemporaldementia (FTD), and many of these cases have ubiquitin-positive,tau-negative inclusions in neurons of the dentate gyms and superficiallayers of the frontal and temporal lobes.

Inclusion-body myositis (IBM) is a crippling disease usually found inpeople over age 50, in which muscle fibers develop inflammation andbegin to atrophy—but in which the brain is spared and patients retaintheir full intellect. Two enzymes involved in the production ofamyloid-β protein were found to be increased inside the muscle cells ofpatients with this most common, progressive muscle disease of olderpeople, in which amyloid-β is also increased.

Another disease that is based on or associated with the accumulation anddeposit of amyloid-like protein is macular degeneration.

Macular degeneration is a common eye disease that causes deteriorationof the macula, which is the central area of the retina (the paper-thintissue at the back of the eye where light-sensitive cells send visualsignals to the brain). Sharp, clear, ‘straight ahead’ vision isprocessed by the macula. Damage to the macula results in the developmentof blind spots and blurred or distorted vision. Age-related maculardegeneration (AMD) is a major cause of visual impairment in the UnitedStates and for people over age 65 it is the leading cause of legalblindness among Caucasians. Approximately 1.8 million Americans age 40and older have advanced AMD, and another 7.3 million people withintermediate AMD are at substantial risk for vision loss. The governmentestimates that by 2020 there will be 2.9 million people with advancedAMD. Victims of AMD are often surprised and frustrated to find out howlittle is known about the causes and treatment of this blindingcondition.

There are two forms of macular degeneration: dry macular degenerationand wet macular degeneration. The dry form, in which the cells of themacula slowly begin to break down, is diagnosed in 85 percent of maculardegeneration cases. Both eyes are usually affected by dry AMD, althoughone eye can lose vision while the other eye remains unaffected. Drusen,which are yellow deposits under the retina, are common early signs ofdry AMD. The risk of developing advanced dry AMD or wet AMD increases asthe number or size of the drusen increases. It is possible for dry AMDto advance and cause loss of vision without turning into the wet form ofthe disease; however, it is also possible for early-stage dry AMD tosuddenly change into the wet form.

The wet form, although it only accounts for 15 percent of the cases,results in 90 percent of the blindness, and is considered advanced AMD(there is no early or intermediate stage of wet AMD). Wet AMD is alwayspreceded by the dry form of the disease. As the dry form worsens, somepeople begin to have abnormal blood vessels growing behind the macula.These vessels are very fragile and will leak fluid and blood (hence‘wet’ macular degeneration), causing rapid damage to the macula.

The dry form of AMD will initially often cause slightly blurred vision.The center of vision in particular may then become blurred and thisregion grows larger as the disease progresses. No symptoms may benoticed if only one eye is affected. In wet AMD, straight lines mayappear wavy and central vision loss can occur rapidly.

Diagnosis of macular degeneration typically involves a dilated eye exam,visual acuity test, and a viewing of the back of the eye using aprocedure called fundoscopy to help diagnose AMD, and—if wet AMD issuspected—fluorescein angiography may also be performed. If dry AMDreaches the advanced stages, there is no current treatment to preventvision loss. However, a specific high dose formula of antioxidants andzinc may delay or prevent intermediate AMD from progressing to theadvanced stage. Macugen® (pegaptanib sodium injection), laserphotocoagulation and photodynamic therapy can control the abnormal bloodvessel growth and bleeding in the macula, which is helpful for somepeople who have wet AMD; however, vision that is already lost will notbe restored by these techniques. If vision is already lost, low visionaids exist that can help improve the quality of life.

One of the earliest signs of age-related macular degeneration (AMD) isthe accumulation of drusen between the basal lamina of the retinalpigmented epithelium (RPE) and Bruch's membrane (BM). Recent studiesconducted by Anderson et al. have confirmed that drusen contains amyloidbeta. (Experimental Eye Research 78 (2004) 243-256).

Ongoing research continues with studies exploring environmental,genetic, and dietary factors that may contribute to AMD. New treatmentstrategies are also being explored, including retinal cell transplants,drugs that will prevent or slow down the progress of the disease,radiation therapy, gene therapies, a computer chip implanted in theretina that may help stimulate vision and agents that will prevent thegrowth of new blood vessels under the macula.

An important factor to consider when developing new drugs is the ease ofuse for the target patients. Oral drug delivery, specifically tablets,capsules and softgels-, account for 70% of all dosage forms consumedbecause of patient convenience. Drug developers agree that patientsprefer oral delivery rather than subjecting themselves to injections orother, more invasive forms of medicinal administration. Formulationsresulting in low dosing intervals (i.e. once a day or sustained release)are also preferable. The ease of administering antibiotics in oraldosage forms results in an increase of patient compliance duringtreatment.

What is needed are effective methods and compositions for the generationof highly specific and highly effective antibodies, which is aprerequisite if the antibodies are to be provided in an oral dosageform. Preferably such antibodies would recognize specific epitopes onvarious antigens such as amyloid protein.

What is also needed therefore, are effective compositions and methodsfor addressing the complications associated with diseases and disorderswhich are caused by or associated with amyloid or amyloid-like proteinsincluding amyloidosis, a group of diseases and disorders associated withamyloid plaque formation including secondary amyloidosis and age-relatedamyloidosis including, but not limited to, neurological disorders suchas Alzheimer's Disease (AD), including diseases or conditionscharacterized by a loss of cognitive memory capacity such as, forexample, mild cognitive impairment (MCI), Lewy body dementia, Down'ssyndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type);the Guam Parkinson-Dementia complex; as well as other diseases which arebased on or associated with amyloid-like proteins such as progressivesupranuclear palsy, multiple sclerosis; Creutzfeld Jacob disease,Parkinson's disease, HIV-related dementia, ALS (amyotropic lateralsclerosis), inclusion-body myositis (IBM), Adult Onset Diabetes; senilecardiac amyloidosis; endocrine tumors, and others, including maculardegeneration. In particular what is need are specialized and highlyeffective antibodies capable of counteracting the physiologicalmanifestations of the disease such as the formation of plaquesassociated with aggregation of fibers of the amyloid or amyloid-likepeptide.

Anti-amyloid antibodies elicited by the inoculation of Aβ₁₋₄₂ mixed withFreund complete or incomplete adjuvant had proved capable to reduce theamyloid burden in transgenic mice for human Alzheimer disease (Schenk etal., 1999).

Intraperitonal inoculation of tetrapalmitoylated Aβ₁₋₁₆ reconstituted inliposomes to NORBA transgenic mice elicited significant titers ofanti-amyloid antibodies, which also proved capable to solubilize amyloidfibers and plaques in vitro and in vivo. (Nicolau et al., 2002).

A possible mechanism by which the dissolution of amyloid plaques andfibres occurred was first suggested by Bard et al., (2000), who advancedthe conclusion, based upon their data, that the antibodies opsonized theplaques, which were subsequently destroyed by the macrophages of themicroglia. De Mattos et al., (2001) indicated that a MAb directedagainst the central domain of β-amyloid was able to bind and completelysequester plasma amyloid. They argued that the presence of these mAbs incirculation shifted the equilibrium of Aβ between brain and plasma,favoring the peripheral clearing and catabolism instead of depositionwithin the brain.

The present invention provides novel methods and compositions comprisinghighly specific and highly effective antibodies having the ability tospecifically recognize and bind to specific β-amyloid proteins. Theantibodies enabled by the teaching of the present invention areparticularly useful for the treatment of diseases and disorders in asubject, which are caused by or associated with amyloid or amyloid-likeproteins including amyloidosis, a group of diseases and disordersassociated with amyloid plaque formation including secondary amyloidosisand age-related amyloidosis including, but not limited to, neurologicaldisorders such as Alzheimer's Disease (AD), including diseases orconditions characterized by a loss of cognitive memory capacity such as,for example, mild cognitive impairment (MCI), Lewy body dementia, Down'ssyndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type);the Guam Parkinson-Dementia complex; as well as other diseases which arebased on or associated with amyloid-like proteins such as progressivesupranuclear palsy, multiple sclerosis; Creutzfeld Jacob disease,hereditary cerebral hemorrhage with amyloidosis Dutch type, Parkinson'sdisease, HIV-related dementia, ALS (amyotropic lateral sclerosis),inclusion-body myositis (IBM), Adult Onset Diabetes; senile cardiacamyloidosis; endocrine tumors, and others, including maculardegeneration, to name just a few.

Moreover, the present invention provides novel methods and compositionsfor retaining or increasing the cognitive memory capacity in a subject,particularly a mammal exhibiting an amyloid-associated disease orcondition comprising administering to a subject, particularly a mammal,more particularly a human in need of such a treatment, a therapeuticallyeffective amount of a monoclonal antibody according to the invention.

SUMMARY OF THE INVENTION

The present invention makes use of antigen presentations that result inenhanced exposure and stabilization of a preferred antigen conformation,which ultimately results in antibodies with unique properties.

In one embodiment of the invention, an antibody is provided includingany functionally equivalent antibody or functional parts thereof, or,more particularly, a monoclonal antibody including any functionallyequivalent antibody or functional parts thereof, which has been raisedagainst a supramolecular antigenic construct comprising an antigenicpeptide corresponding to the amino acid sequence of the β-amyloidpeptide, particularly of β-amyloid peptide Aβ₂₂₋₃₅ and Aβ₂₉₋₄₀,respectively, modified with a hydrophilic moiety such as, for example,polyethylene glycol (PEG), wherein said hydrophilic moiety, iscovalently bound to each of the termini of the antigenic peptide throughat least one, particularly one or two amino acids such as, for example,lysine, glutamic acid and cysteine or any other suitable amino acid oramino acid analogue capable of serving as a connecting device forcoupling the hydrophilic moiety to the peptide fragment. When a PEG isused as the hydrophilic moiety, the free PEG termini may be covalentlybound to phosphatidylethanolamine or any other compound suitable tofunction as the anchoring element, for example to embed the antigenicconstruct in the bilayer of a liposome.

In one embodiment of the invention, an antibody is provided,particularly a monoclonal antibody including any functionally equivalentantibody or functional parts thereof, which antibody recognizes andbinds to a conformational epitope and binds to polymeric soluble amyloidand to amyloid fibrils or fibers, respectively, particularly topolymeric soluble amyloid Aβ peptides and amyloid Aβ fibrils or fibers,respectively.

In one embodiment of the invention, an antibody is provided,particularly a monoclonal antibody including any functionally equivalentantibody or functional parts thereof, which antibody recognizes andbinds to a conformational epitope preferentially displayed on polymericsoluble amyloid and oligomeric amyloid peptides, respectively, but alsoon amyloid fibrils or fibers, particularly on polymeric soluble amyloidAβ peptides and oligomeric amyloid Aβ peptides comprising a plurality ofmonomeric Aβ 1-42 peptides, and on amyloid fibrils or fibersincorporating a plurality of said oligomeric peptides, respectively.

The inheritance of the ε4 allele of the apolipoproteinE (apoE4) proteinis a strong genetic risk factor for AD. This protein is able to bind toamyloid and is known to be involved in both the clearance of Aβ acrossthe blood-brain-barrier as well as the promotion of Aβ deposition. Inreverse, the binding of amyloid maps in the hydrophobic lipoproteinbinding region of ApoE and this association dramatically diminish theoverall lipid binding ability of ApoE.

Accordingly, it is a further embodiment of the invention to provide anantibody, particularly a monoclonal antibody, including any functionallyequivalent antibody or functional parts thereof as described herein,which antibody is capable of inhibiting or otherwise lessening theinteraction of amyloid with ApoE4 in the brain of a subject,particularly a mammal, but especially a human, particularly in the brainof a subject, particularly a mammal, but especially a human sufferingfrom a disease or condition associated with increased concentration ofAβ in the brain.

Accordingly, it is a further embodiment of the invention to provide anantibody, particularly a monoclonal antibody, including any functionallyequivalent antibody or functional parts thereof as described herein,which antibody is capable of inhibiting or otherwise lessening theinteraction of amyloid with ApoE4 in the brain of a subject,particularly a mammal, but especially a human, particularly in the brainof a subject, particularly a mammal, but especially a human sufferingfrom a disease or condition associated with increased concentration ofAβ in the brain. The antibody according to the present invention,particularly a monoclonal antibody including any functionally equivalentantibody or functional parts thereof, thus preferentially binds topolymeric soluble amyloid and amyloid fibrils or fibers, respectively,particularly to polymeric soluble Aβ peptides comprising a plurality ofAβ₁₋₄₂ monomeric peptides and Aβ fibrils or fibers incorporating aplurality of said polymeric peptides, respectively.

In one embodiment the invention relates to an antibody, as describedherein, particularly a monoclonal antibody including any functionallyequivalent antibody or functional parts thereof, which antibody binds toAβ monomeric peptides having at least 10, particularly at least 20, moreparticularly at least 30, even more particularly at least 40 amino acidresidues, and/or to soluble polymeric amyloid peptides comprising aplurality of said amyloid monomeric peptides and/or to amyloid fibers orfibrils incorporating a plurality of said polymeric peptides butespecially to an Aβ₁₋₄₂ monomeric peptide and polymeric soluble Aβpeptides comprising a plurality of Aβ₁₋₄₂ monomeric peptides and Aβfibrils or fibers incorporating a plurality of said polymeric peptides,and shows essentially no binding to Aβ monomeric peptides having 8 orfewer amino acid residues.

In one specific embodiment, the invention relates to an antibody asdescribed herein, particularly a monoclonal antibody including anyfunctionally equivalent antibody or functional parts thereof, whichantibody binds to Aβ monomeric peptides having at least 30, even moreparticularly at least 40 amino acid residues, and/or to solublepolymeric amyloid peptides comprising a plurality of said amyloidmonomeric peptides and/or to amyloid fibers or fibrils incorporating aplurality of said polymeric peptides but especially to an Aβ₁₋₄₂monomeric peptide and polymeric soluble Aβ peptides comprising aplurality of β₁₋₄₂ monomeric peptides and Aβ fibrils or fibersincorporating a plurality of said polymeric peptides, but showsessentially no binding to Aβ monomeric peptide 17-40.

In another specific embodiment of the invention, an antibody is providedas described herein, particularly a monoclonal antibody including anyfunctionally equivalent antibody or functional parts thereof, whichantibody binds to Aβ monomeric peptide 1-42 and polymeric soluble Aβpeptides comprising a plurality of Aβ₁₋₄₂ monomeric peptides and Aβfibrils or fibers incorporating a plurality of said polymeric peptides,but shows a substantially weaker binding to Aβ monomeric peptide 1-28.

In another specific embodiment of the invention, an antibody is providedas described herein, particularly a monoclonal antibody including anyfunctionally equivalent antibody or functional parts thereof, whichantibody binds to Aβ monomeric peptide 1-42 and polymeric soluble Aβpeptides comprising a plurality of Aβ₁₋₄₂ monomeric peptides and Aβfibrils or fibers incorporating a plurality of said polymeric peptides,but shows a substantially weaker binding to Aβ monomeric peptide 1-28and essentially no binding to Aβ monomeric peptide 17-40.

By a “substantially weaker binding” a binding is meant, which is atleast about 60%, particularly at least about 65%, more particularly atleast about 70%, even more particularly at least about 80%, butespecially at least about 90% and up to 100% less than the binding to Aβmonomeric peptide 1-42.

By “essentially no binding” a binding is meant, which is at least about85%, particularly at least about 90%, more particularly at least about95%, even more particularly at least about 98%, but especially at leastabout 99% and up to 100% less than the binding to Aβ monomeric peptide1-42.

In one embodiment of the invention the binding of the antibody accordingto the invention as described herein, particularly a monoclonal antibodyincluding any functionally equivalent antibody or functional partsthereof, to Aβ monomeric peptides is determined by an ELISA-type assay,particularly by an ELISA assay using biotinylated Aβ monomeric peptides,but especially by an ELISA assay as described in Example 13 below.

In one embodiment the antibody according to the invention and asdescribed herein, upon co-incubation with an Aβ peptide in a monomericand/or an oligomeric form having at least 30, particularly at least 35,more particularly at least 38, even more particularly at least 40 aminoacid residues, but especially with an Aβ₁₋₄₂ peptide, particularly at amolar concentration ratio of antibody to Aβ₁₋₄₂ of up to 1:1000, butespecially at a molar concentration ratio of between 1:10 and 1:100,inhibits the aggregation of the Aβ monomers and/or oligomers to highmolecular polymeric fibrils.

In particular, the co-incubation of the antibody according to theinvention with amyloid monomeric and/or oligomeric peptides is carriedout for 24 hours to 60 hours, particularly for 30 hours to 50 hours,more particularly for 48 hours at a temperature of between 28° C. and40° C., particularly of between 32° C. and 38° C., more particularly at37° C.

In a specific embodiment of the invention, co-incubation with amyloidmonomeric and/or oligomeric peptides is accomplished for 48 hours at atemperature of 37° C.

In particular, the antibody, particularly a monoclonal antibodyaccording to the invention including any functionally equivalentantibody or functional parts thereof binds to Aβ1-42 monomeric peptideand polymeric soluble Aβ peptides comprising a plurality of Aβ1-42monomeric peptides and Aβ fibrils or fibers incorporating a plurality ofsaid polymeric peptides, but shows a substantially weaker binding to Aβmonomeric peptide 1-28 and/or essentially no binding to Aβ monomericpeptide 17-40. and, upon co-incubation with Aβ₁₋₄₂ monomeric and/oroligomeric peptide inhibits the aggregation of the Aβ monomers to highmolecular polymeric fibrils.

In one embodiment, the antibody, particularly a monoclonal antibodyaccording to the invention including any functionally equivalentantibody or functional parts thereof inhibits the aggregation of the Aβmonomers and/or oligomers to high molecular polymeric fibrils by atleast 20%, by at least 30%, particularly by at least 40%, particularlyby at least 50%, more particularly by at least 60%, even moreparticularly by at least 70%, but especially by at least 80%-90%, ormore as compared to the respective amyloid peptide monomers incubated inbuffer (control).

In one embodiment of the invention, an antibody is provided,particularly a monoclonal antibody according to the invention includingany functionally equivalent antibody or functional parts thereof, whichantibody binds to Aβ1-42 monomeric peptide and polymeric soluble Aβpeptides comprising a plurality of Aβ1-42 monomeric peptides and Aβfibrils or fibers incorporating a plurality of said polymeric peptides,but shows a substantially weaker binding to Aβ monomeric peptide 1-28and/or essentially no binding to Aβ monomeric peptide 17-40 and, uponco-incubation with Aβ₁₋₄₂ monomeric and/or oligomeric peptide for 24hours at a temperature of 37° C. inhibits the aggregation of the Aβmonomers to high molecular polymeric fibrils by at least 20%, by atleast 30%, particularly by at least 40%, particularly by at least 50%,more particularly by at least 60%, even more particularly by at least70% at a molar concentration ratio of antibody to Aβ₁₋₄₂ of 1:100 and byat least 50%, more particularly by at least 60%, more particularly by atleast 65%, even more particularly by at least 70% at a molarconcentration ratio of antibody to Aβ₁₋₄₂ of 1:10 as determined by athioflavin T (Th-T) fluorescent assay, particularly a thioflavin T(Th-T) fluorescent assay as described in Example 4.

Binding of the antibodies according to the invention and as describedherein to amyloidogenic monomeric and/or oligomeric peptides but,particularly, to the amyloid form (1-42) leads to inhibition of theaggregation of monomeric and/or oligomeric amyloidogenic peptides tohigh molecular fibrils or filaments. Through the inhibition of theaggregation of amyloidogenic monomeric and/or oligomeric peptides theantibodies according to the present invention are capable of preventingor slowing down the formation of amyloid plaques, particularly theamyloid form (1-42), which is known to become insoluble by a change ofsecondary conformation and to be the major part of amyloid plaques inbrains of diseased animals or humans.

The aggregation inhibition potential of the antibody according to theinvention may be determined by any suitable method known in the art, forexample, by density-gradient ultracentrifugation followed by a SDS-PAGEsedimentation analysis on a preformed gradient and/or by a thioflavin T(Th-T) fluorescent assay.

In one embodiment, the invention relates to an antibody, particularly amonoclonal antibody as described herein including any functionallyequivalent antibody or functional parts thereof, which antibody, uponco-incubation, particularly at a molar concentration ratio of between1:10 and 1:1000, more particularly at a ratio of between 1:10 and 1:100,with preformed high molecular polymeric amyloid fibrils or filamentsformed by the aggregation of Aβ monomeric and/or oligomeric peptideshaving at least 30, particularly at least 35, more particularly at least38, even more particularly at least 40 amino acid residues and, butespecially of Aβ₁₋₄₂ monomeric and/or oligomeric peptides, is capable ofdisaggregating the preformed polymeric fibrils or filaments by at least10%, by at least 20%, particularly by at least 30%, more particularly byat least 40%, even more particularly by at least 50%, but especially byat least 60%-70% or more.

In a specific embodiment of the invention, the aggregation inhibitionand the disaggregation potential of the antibody, respectively, aredetermined by density-gradient ultracentrifugation followed by aSDS-PAGE sedimentation analysis on a preformed gradient.

In another specific embodiment of the invention, the aggregationinhibition and the disaggregation potential of the antibody,respectively, are determined by a thioflavin T (Th-T) fluorescent assay.

In another specific embodiment, the antibody according to the inventionis co-incubated with preformed high molecular polymeric amyloid fibrilsor filaments for 12 hours to 36 hours, particularly for 18 hours to 30hours, more particularly for 24 hours at a temperature of between 28° C.and 40° C., particularly of between 32° C. and 38° C., more particularlyat 37° C.

In particular, the co-incubation with preformed high molecular polymericamyloid fibrils or filaments is performed for 24 hours at a temperatureof 37° C.

In particular, the invention relates to an antibody, particularly themonoclonal antibody according to the invention including anyfunctionally equivalent antibody or functional parts thereof, whichantibody binds to Aβ1-42 monomeric peptide and polymeric soluble Aβpeptides comprising a plurality of Aβ1-42 monomeric peptides and Aβfibrils or fibers incorporating a plurality of said polymeric peptides,but shows a substantially weaker binding to Aβ monomeric peptide 1-28and/or essentially no binding to Aβ monomeric peptide 17-40. and, uponco-incubation with preformed high molecular polymeric amyloid fibrils orfilaments formed by the aggregation of Aβ₁₋₄₂ monomeric and/oroligomeric peptides is capable of disaggregating the preformed polymericfibrils or filaments, particularly by at least 10%, by at least 20%,particularly by at least 30%, more particularly by at least 40%, evenmore particularly by at least 50%, but especially by at least 60% ormore.

In one embodiment of the invention, an antibody is provided,particularly a monoclonal antibody according to the invention includingany functionally equivalent antibody or functional parts thereof, whichantibody binds to Aβ1-42 monomeric peptide and polymeric soluble Aβpeptides comprising a plurality of Aβ1-42 monomeric peptides and Aβfibrils or fibers incorporating a plurality of said polymeric peptides,but shows a substantially weaker binding to Aβ monomeric peptide 1-28and/or essentially no binding to Aβ monomeric peptide 17-40 and, uponco-incubation with preformed high molecular polymeric amyloid fibrils orfilaments formed by the aggregation of Aβ₁₋₄₂ monomeric and/oroligomeric peptide for 24 hours at a temperature of 37° C. results in adisaggregation of the preformed polymeric fibrils or filaments by atleast 10%, by at least 20%, by at least 30%, particularly by at least40%, particularly by at least 50%, more particularly by at least 60%,even more particularly by at least 70%, at a molar concentration ratioof antibody to Aβ₁₋₄₂ of 1:100 and by at least 40%, particularly by atleast 50%, more particularly by at least 60%, even more particularly byat least 70% at a molar concentration ratio of antibody to Aβ₁₋₄₂ of1:10 as determined by a thioflavin T (Th-T) fluorescent assay,particularly a thioflavin T (Th-T) fluorescent assay as described inExample 4 below.

Through both the inhibition of the aggregation of amyloid protein andthrough the disaggregation of amyloidogenic polymeric fibrils orfilaments the antibodies according to the present invention are capableof preventing or slowing down the formation of amyloid plaques whichleads to an alleviation of the symptoms associated with the disease anda delay or reversal of its progression.

Accordingly, it is a further embodiment of the invention to provide anantibody, particularly a monoclonal antibody, including any functionallyequivalent antibody or functional parts thereof as described herein,which antibody is capable of decreasing the total amount of Aβ in thebrain of a subject, particularly a mammal, but especially a humansuffering from a disease or condition associated with to increasedconcentration of Aβ in the brain.

In another embodiment of the invention an antibody, particularly amonoclonal antibody, including any functionally equivalent antibody orfunctional parts thereof as described herein is provided, which antibodyis capable of disrupting plaques thus decreasing the plaque load in thebrain of a subject, particularly a mammal, but especially a humansuffering from a disease or condition associated with an increasedplaque load in the brain. An antibody according to the inventionincluding any functionally equivalent antibody or functional partsthereof decreases the plaque load in the brain by at least 20%,particularly by at least 25%, more particularly by at least 30%, evenmore particularly more than 30%.

In still another embodiment of the invention an antibody, particularly amonoclonal antibody, including any functionally equivalent antibody orfunctional parts thereof as described herein is provided, which antibodyis capable of solubilizing plaques associated with a reduction of theamount of plaques in the brain of a subject, particularly a mammal, butespecially a human suffering from a disease or condition associated withan increased plaque load in the brain. An antibody according to theinvention including any functionally equivalent antibody or functionalparts thereof reduces the amount of plaques in the brain by at least10%, particularly by at least 15%, more particularly by at least 20%.

It is to be understood that an antibody according to the invention canexhibit one, two or more of the specific properties described herein invarious combinations.

For example, in one embodiment, the present invention providesantibodies, but especially monoclonal antibodies including anyfunctionally equivalent antibody or functional parts thereof, whichantibodies are bi-specific or bi-effective in that they exhibit both anaggregation inhibition property as well as a disaggregation property asdefined herein, particularly paired with a high degree of conformationalsensitivity.

In one embodiment, an antibody according to the invention and asdescribed herein is bi-specific or bi-effective and, upon co-incubationwith an Aβ monomeric and/or oligomeric peptide having at least 30,particularly at least 35, more particularly at least 38, even moreparticularly at least 40 amino acid residues, but especially with anAβ₁₋₄₂ monomeric and/or oligomeric peptide, inhibits the aggregation ofthe Aβ monomers to high molecular polymeric fibrils and, in addition,upon co-incubation with preformed high molecular polymeric amyloidfibrils or filaments formed by the aggregation of Aβ monomeric and/oroligomeric peptides having at least 30, particularly at least 35, moreparticularly at least 38, even more particularly at least 40 amino acidresidues and, but especially Aβ₁₋₄₂ monomeric and/or oligomericpeptides, is capable of disaggregating the preformed polymeric fibrilsor filaments.

In particular, co-incubation with amyloid monomeric and/or oligomericpeptides and preformed high molecular polymeric amyloid fibrils orfilaments, respectively, takes place at a molar concentration ratio ofup to 1:1000, but especially at a molar concentration ratio of between1:10 and 1:1000, particularly at a molar concentration ratio of 1:100.

Co-incubation of an antibody according to the invention with amyloidmonomeric and/or oligomeric peptides is carried out for 24 hours to 60hours, particularly for 30 hours to 50 hours, more particularly for 48hours at a temperature of between 28° C. and 40° C., particularly ofbetween 32° C. and 38° C., more particularly at 37° C., whereas theco-incubation with amyloid preformed high molecular polymeric amyloidfibrils or filaments is carried out for 12 hours to 36 hours,particularly for 18 hours to 30 hours, more particularly for 24 hours ata temperature of between 28° C. and 40° C., particularly of between 32°C. and 38° C., more particularly at 37° C.

In one embodiment, a bi-specific or bi-effective antibody according tothe invention and as described herein, particularly a monoclonalantibody including any functionally equivalent antibody or functionalparts thereof, is capable of disaggregating the preformed polymericfibrils or filaments by at least 10%, by at least 20%, by at least 30%,particularly by at least 40%, particularly by at least 50%, moreparticularly by at least 60%, even more particularly by at least 70%.

In one embodiment, a bi-specific or bi-effective antibody according tothe invention and as described herein, particularly a monoclonalantibody including any functionally equivalent antibody or functionalparts thereof, is capable of inhibiting the aggregation of Aβ monomericand/or oligomeric peptides having at least 30, particularly at least 35,more particularly at least 38, even more particularly at least 40 aminoacid residues, but especially Aβ₁₋₄₂ monomeric and/or oligomericpeptides by at least 30%, particularly by at least 40%, particularly byat least 50%, more particularly by at least 60%, even more particularlyby at least 70%, but especially by at least 80-90%, or more as comparedto the respective amyloid peptide monomers incubated in buffer(control).

In one embodiment, the invention provides a bi-specific or bi-effectiveantibody as described herein, particularly a monoclonal antibodyincluding any functionally equivalent antibody or functional partsthereof, which antibody exhibits high specificity to Aβ₁₋₄₂ monomericpeptides but shows essentially no or only minor cross-reactivity to anamyloid peptide monomer selected from the group consisting of Aβ₁₋₂₈,and M₁₇₋₄₀ monomeric peptides.

In a specific embodiment, the invention relates to an antibody asdescribed herein, particularly a monoclonal antibody including anyfunctionally equivalent antibody or functional parts thereof, whichantibody is up to 1000 fold, particularly 50 to 1000 fold, moreparticularly 80 to 100 fold, but especially 100 fold more sensitive toamyloid peptide Aβ₁₋₄₂ as compared to Aβ₁₋₂₈, and/or Aβ₁₇₋₄₀, and iscapable of inhibiting, in vitro and in vivo, the aggregation ofamyloidogenic monomeric and/or oligomeric peptides and/or ofdisaggregating preformed polymeric fibrils or filaments.

In one embodiment, the invention provides a bi-specific or bi-effectiveantibody as described herein, particularly a monoclonal antibodyincluding any functionally equivalent antibody or functional partsthereof, which antibody binds to Aβ1-42 monomeric peptide and polymericsoluble Aβ peptides comprising a plurality of Aβ1-42 monomeric peptidesand Aβ fibrils or fibers incorporating a plurality of said polymericpeptides, but shows a substantially weaker binding to Aβ monomericpeptide 1-28 and/or essentially no binding to Aβ monomeric peptide 17-40and, upon co-incubation with Aβ₁₋₄₂ monomeric and/or oligomeric peptidefor 24 hours at a temperature of 37° C. inhibits the aggregation of theAβ monomers to high molecular polymeric fibrils by at least 30% at amolar concentration ratio of antibody to Aβ₁₋₄₂ of 1:100 and by at least65% at a molar concentration ratio of antibody to Aβ₁₋₄₂ of 1:10 and,upon co-incubation with preformed high molecular polymeric amyloidfibrils or filaments formed by the aggregation of Aβ₁₋₄₂ monomericand/or oligomeric peptide for 24 hours at a temperature of 37° C.results in a disaggregation of the preformed polymeric fibrils orfilaments by at least 20%, at a molar concentration ratio of antibody toAβ₁₋₄₂ of 1:100 and by at least 50% at a molar concentration ratio ofantibody to Aβ₁₋₄₂ of 1:10 as determined by a thioflavin T (Th-T)fluorescent assay, particularly a thioflavin T (Th-T) fluorescent assayas described in Example 4 below.

In another specific embodiment, the invention relates to an antibody asdescribed herein, particularly a monoclonal antibody including anyfunctionally equivalent antibody or functional parts thereof, whichantibody has a high binding sensitivity to amyloid peptide Aβ₁₋₄₂ and iscapable of detecting Aβ₁₋₄₂ fibers in a concentration of up to 0.01 μg,but particularly in a concentration range of between 0.5 μg and 0.01 μg,more particularly between 0.1 μg and 0.01 μg, but especially in aconcentration of 0.01 μg.

In one embodiment, the invention provides an antibody as describedherein, particularly a monoclonal antibody including any functionallyequivalent antibody or functional parts thereof, which antibody has beenraised against a supramolecular antigenic construct comprising anantigenic peptide corresponding to the amino acid sequence of theβ-amyloid peptide Aβ₂₂₋₃₅ and Aβ₂₉₋₄₀, respectively, modified with ahydrophilic moiety such as, for example, polyethylene glycol (PEG),wherein said hydrophilic moiety is covalently bound to each terminusthrough an amino acid such as, for example, lysine or any other suitableamino acid or amino acid analogue capable of serving as a linkermolecule.

The antibody according to the invention and as described herein,particularly a monoclonal antibody including any functionally equivalentantibody or functional parts thereof recognizes and binds to aconformational epitope.

In one embodiment, the invention relates to a light chain variableregion exhibiting an amino acid sequence that is 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to thesequences given in SEQ ID NO: 7 and SEQ ID NO: 9, respectively, or afunctional part thereof comprising at least one, particularly at leasttwo, more particularly at least 3 of the light chain CDRs, butespecially all CDRs embedded in their natural framework regions.

In one embodiment, the invention relates to a heavy chain variableregion exhibiting an amino acid sequence that is 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to thesequences given in SEQ ID NO: 8 and SEQ ID NO: 10, respectively, or afunctional part thereof comprising at least one, particularly at leasttwo, more particularly at least 3 of the heavy chain CDRs, butespecially all CDRs embedded in their natural framework regions.

Further, the invention relates to an antibody, particularly a monoclonalantibody including any functionally equivalent antibody or functionalparts thereof according to the present invention and as described hereinwherein said antibody comprises a light chain variable domain exhibitingan amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences given inSEQ ID NO: 7 and SEQ ID NO: 9, respectively, or a functional partthereof comprising at least one, particularly at least two, moreparticularly at least 3 of the light chain CDRs, but especially all CDRsembedded in their natural framework regions.

Further, the invention relates to an antibody, particularly a monoclonalantibody including any functionally equivalent antibody or functionalparts thereof according to the present invention and as described hereinwherein said antibody comprises a heavy chain variable domain exhibitingan amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences given inSEQ ID NO: 8 and SEQ ID NO: 10, respectively, or a functional partthereof comprising at least one, particularly at least two, moreparticularly at least 3 of the heavy chain CDRs, but especially all CDRsembedded in their natural framework regions.

In one embodiment, the invention relates to an antibody, particularly amonoclonal antibody including any functionally equivalent antibody orfunctional parts thereof according to the present invention and asdescribed herein, wherein said antibody comprises a light chain and aheavy chain variable domain exhibiting an amino acid sequence that is85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the sequences given in SEQ ID NO: 7, SEQ ID NO: 9, SEQID NO: 8 and SEQ ID NO: 10, or a functional part thereof comprising partor all the heavy and the light chain CDRs.

The invention further relates to a monoclonal antibody including anyfunctionally equivalent antibody or functional parts thereof whichantibody comprises the polypeptide sequence as given in SEQ ID NOs: 7and SEQ ID NOs:9 and/or in SEQ ID NO: 8 and SEQ ID NOs:10. The inventionfurther relates to the monoclonal antibody ACI-11-Ab-9 having thepolypeptide sequences SEQ ID NO: 7-8 and ACI-12-Ab-11 having thepolypeptide sequences SEQ ID NOs: 9-10.

Also comprised by the present invention is an antibody the sequence ofwhich has been altered by introducing at least one, particularly atleast two, more particularly at least 3 or more conservativesubstitutions into the sequences of SEQ ID NOs: 7-8 and SEQ ID NOs:9-10, respectively, such that the antibody essentially maintains itsfull functionality.

In one embodiment the invention relates to a peptide fragment comprisingthe light chain CDR1 as given in SEQ ID NO:11 and/or the light chainCDR2 as given in SEQ ID NO:12 and/or the light chain CDR3 as given inSEQ ID NO:13.

In one embodiment the invention relates to a peptide fragment comprisingthe heavy chain CDR1 as given in SEQ ID NO:14 and/or the heavy chainCDR2 as given in SEQ ID NO:15 and/or the heavy chain CDR3 as given inSEQ ID NO:16.

In one embodiment the invention relates to the light chain CDR1 as givenin SEQ ID NO:11.

In one embodiment the invention relates to the light chain CDR2 as givenin SEQ ID NO:12.

In one embodiment the invention relates to the light chain CDR3 as givenin SEQ ID NO:13.

In one embodiment the invention relates to the heavy chain CDR1 as givenin SEQ ID NO:14.

In one embodiment the invention relates to the heavy chain CDR2 as givenin SEQ ID NO:15.

In one embodiment the invention relates to the heavy chain CDR3 as givenin SEQ ID NO:16.

In one embodiment, the invention relates to a polynucleotide comprisinga nucleotide sequence encoding a light chain variable region exhibitingan amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences given inSEQ ID NO: 7 and SEQ ID NO: 9, respectively, or a functional partthereof comprising at least one, particularly at least two, moreparticularly at least 3 of the light chain CDRs as depicted in SEQ IDNOs: 11-13, but especially all CDRs embedded in their natural frameworkregions.

In one embodiment, the invention relates to a polynucleotide comprisinga nucleotide sequence encoding a heavy chain variable region exhibitingan amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences given inSEQ ID NO: 8 and SEQ ID NO: 10, respectively, or a functional partthereof comprising at least one, particularly at least two, moreparticularly at least 3 of the heavy chain CDRs as depicted in SEQ IDNOs: 14-16, but especially all CDRs embedded in their natural frameworkregions.

In one embodiment, the invention relates to a polynucleotide comprisinga nucleotide sequence encoding an antibody, particularly a monoclonalantibody including any functionally equivalent antibody or functionalparts thereof according to the present invention and as described hereinwherein said antibody comprises a light chain variable domain exhibitingan amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences given inSEQ ID NO: 7 and SEQ ID NO: 9, respectively, or a functional partthereof comprising at least one, particularly at least two, moreparticularly at least 3 of the light chain CDRs as depicted in SEQ IDNOs: 11-13, but especially all CDRs embedded in their natural frameworkregions.

In one embodiment, the invention relates to a polynucleotide comprisinga nucleotide sequence encoding an antibody, particularly a monoclonalantibody including any functionally equivalent antibody or functionalparts thereof according to the present invention and as described hereinwherein said antibody comprises a heavy chain variable domain exhibitingan amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequences given inSEQ ID NO: 8 and SEQ ID NO: 10, respectively, or a functional partthereof comprising at least one, particularly at least two, moreparticularly at least 3 of the heavy chain CDRs as depicted in SEQ IDNOs: 14-16, but especially all CDRs embedded in their natural frameworkregions.

In one embodiment, the invention relates to a polynucleotide comprisinga nucleotide sequence encoding an antibody, particularly a monoclonalantibody including any functionally equivalent antibody or functionalparts thereof according to the present invention and as described hereinwherein said antibody comprises a light chain and a heavy chain variabledomain exhibiting an amino acid sequence that is 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to thesequences given SEQ ID NO: 7 and SEQ ID NO: 9 and in SEQ ID NO: 8 andSEQ ID NO: 10, respectively, or a functional part thereof comprising thelight chain and the heavy chain CDRs as depicted in SEQ ID NOs: 11-16.

In another embodiment of the invention, a polynucleotide is providedcomprising a nucleotide sequence encoding the antibody according to theinvention as described herein, but particularly a nucleotide sequenceencoding a monoclonal antibody having the polypeptide sequences in SEQID NOs: 7-8 or a monoclonal antibody having the polypeptide sequencesSEQ ID NOs: 9-10. In particular, the polynucleotides encoding SEQ IDNOs: 7-8 and SEQ ID NOs:9-10 are SEQ ID NOs: 17-18 and SEQ ID NO: 19-20,respectively.

In another embodiment, a polynucleotide is provided which hybridizesunder stringent conditions to a nucleotide sequence encoding themonoclonal antibody having the polypeptide sequences SEQ ID NOs: 7-8 ora monoclonal antibody having the polypeptide sequences SEQ ID NOs: 9-10.In particular a polynucleotide is provided which hybridizes understringent conditions to nucleotides sequences SEQ ID NOs: 17-18 or SEQID NO: 19-20.

In a specific embodiment, the invention relates to a monoclonal antibodyincluding any functionally equivalent antibody or functional partsthereof which antibody has the characteristic properties of an antibodyproduced by hybridoma cell line FG1F9E4, deposited on May 25, 2007 asDSM ACC2845.

In particular, the invention relates to a monoclonal antibody includingany functionally equivalent antibody or functional parts thereofproduced by hybridoma cell line FG1F9E4, deposited on May 25, 2007 asDSM ACC2845.

In a specific embodiment, the invention relates to a monoclonal antibodyincluding any functionally equivalent antibody or functional partsthereof which antibody has the characteristic properties of an antibodyproduced by hybridoma cell line FK2A6A6, deposited on May 25, 2007 asDSM ACC2846.

In particular, the invention relates to a monoclonal antibody includingany functionally equivalent antibody or functional parts thereofproduced by hybridoma cell line FK2A6A6, deposited on May 25, 2007 asDSM ACC2846.

In particular, the invention also relates to an Aβ epitope which bind toa monoclonal antibody including any functionally equivalent antibody orfunctional parts thereof, which antibody has been raised against asupramolecular antigenic construct comprising an antigenic peptidecorresponding to the amino acid sequence of the β-amyloid peptide,Aβ₂₂₋₃₅ and Aβ₂₉₋₄₀, respectively, modified with a hydrophilic moietysuch as, for example, polyethylene glycol (PEG), wherein saidhydrophilic moiety is covalently bound to each terminus through an aminoacid such as, for example, lysine or any other suitable amino acid oramino acid analogue capable of serving as a linker molecule. Theinvention further relates to an Aβ epitope which binds to the monoclonalantibody ACI-11-Ab-9. The invention further relates to an Aβ epitopewhich binds to the monoclonal antibody ACI-12-Ab-11.

In one aspect, an antibody according to the invention as describedherein, particularly a monoclonal antibody including any functionallyequivalent antibody or functional parts thereof is capable of decreasingthe total amount of soluble Aβ in the brain of a subject, particularly amammal, but especially a human suffering from a disease or conditionassociated with increased concentrations of soluble Aβ in the brain.

In another aspect, an antibody according to the invention and asdescribed herein is capable of disrupting plaques thus decreasing theplaque load in the brain of a subject, particularly a mammal, butespecially a human suffering from a disease or condition associated withan increased plaque load in the brain.

In another aspect, an antibody according to the invention and asdescribed herein is capable of solubilizing plaques leading to areduction of the amount of plaques in the brain of a subject,particularly a mammal, but especially a human suffering from a diseaseor condition associated with an increased plaque load in the brain.

It is another object of the present invention to provide methods andcompositions comprising an antibody according to the invention and asdescribed herein for the prevention and/or therapeutic treatment and/oralleviation of the effects of diseases and disorders in a subject, whichare caused by or associated with amyloid or amyloid-like proteinsincluding but not limited to, amyloidosis (a group of diseases anddisorders associated with amyloid plaque formation including secondaryamyloidosis and age-related amyloidosis such as diseases including, butnot limited to, neurological disorders such as Alzheimer's Disease (AD),diseases or conditions characterized by a loss of cognitive memorycapacity such as, for example, mild cognitive impairment (MCI), Lewybody dementia, Down's syndrome, hereditary cerebral hemorrhage withamyloidosis (Dutch type); the Guam Parkinson-Dementia complex; as wellas other diseases and conditions which are based on or associated withamyloid-like proteins such as progressive supranuclear palsy, multiplesclerosis; Creutzfeld Jacob disease, Parkinson's disease, HIV-relateddementia, ALS (amyotropic lateral sclerosis), inclusion-body myositis(IBM), Adult Onset Diabetes; and senile cardiac amyloidosis); endocrinetumors, and macular degeneration, for example, by passively immunizing asubject, including a human or animal, with an antibody according to theinvention and as described herein. In a further aspect of the inventionthe monoclonal antibodies of these methods are ACI-11-Ab-9 having thepolypeptide sequences SEQ ID NOs: 7-8 or ACI-12-Ab-11 having thepolypeptide sequences SEQ ID NO: 9-10, respectively, or a functionalpart thereof as described herein.

The invention further relates to a therapeutic composition comprising anantibody as described herein, particularly a monoclonal antibodyincluding any functionally equivalent antibody or functional partsthereof in a therapeutically effective amount.

In one embodiment, the pharmaceutical composition further comprises apharmaceutically acceptable carrier, particularly in a therapeuticallyeffective amount.

In one embodiment, the invention provides a composition as describedherein for use in the treatment of diseases and disorders in a subject,which are caused by or associated with amyloid or amyloid-like proteinsincluding, but not limited to, amyloidosis, tumors, and maculardegeneration. In a further aspect of such embodiments the monoclonalantibodies used are selected from monoclonal antibody proteinACI-11-Ab-9 having the polypeptide sequences SEQ ID NOs: 7-8 andmonoclonal antibody protein ACI-12-Ab-11 having the polypeptidesequences SEQ ID NO: 9-10, or a functional part thereof.

An antibody, according to the invention, particularly a monoclonalantibody including any functionally equivalent antibody or functionalparts thereof may be administered in combination with other biologicallyactive substances or other treatment procedures for the treatment ofdiseases. The other biologically active substances may be part of thesame composition already comprising an antibody according to theinvention, in the form of a mixture, wherein the antibody and the otherbiologically active substance are intermixed in or with the samepharmaceutically acceptable solvent and/or carrier or the antibody andthe other biologically active substance may be provided separately aspart of a separate composition, which may be offered separately ortogether in the form of a kit of parts.

The antibody, particularly a monoclonal antibody according to theinvention including any functionally equivalent antibody or functionalparts thereof may be administered to a subject in need thereof at thesame time with the other biologically active substance or substances,intermittently or sequentially. For example, a monoclonal antibodyaccording to the invention including any functionally equivalentantibody or functional parts thereof, may be administered to a subjectin need thereof simultaneously with a first additional biologicallyactive substance or sequentially after or before administration of theantibody. If an application scheme is chosen where more than oneadditional biologically active substance are administered together withthe at least one antibody according to the invention, the compounds orsubstances may partially be administered simultaneously, partiallysequentially in various combinations.

In one embodiment, the invention relates to a therapeutic compositioncomprising an antibody as described herein, particularly a monoclonalantibody including any functionally equivalent antibody or functionalparts thereof in a therapeutically effective amount and a furtherbiologically active substance or compound, particularly a compound usedin the medication of diseases and disorders in a subject, which arecaused by or associated with amyloid or amyloid-like proteins including,but not limited to, amyloidosis, endocrine tumors, and maculardegeneration, and/or a pharmaceutically acceptable carrier and/or adiluent and/or an excipient.

In one embodiment, a therapeutic composition is provided according tothe invention and as described herein comprising an antibody asdescribed herein, particularly a monoclonal antibody including anyfunctionally equivalent antibody or functional parts thereof, andfurther comprising at least one compound selected from the groupconsisting of compounds against oxidative stress, anti-apoptoticcompounds, metal chelators, inhibitors of DNA repair such as pirenzepinand metabolites, 3-amino-1-propanesulfonic acid (3APS),1,3-propanedisulfonate (1,3PDS), secretase activators, β- andγ-secretase inhibitors, tau proteins, neurotransmitter, β-sheetbreakers, anti-inflammatory molecules, or cholinesterase inhibitors(ChEIs) such as tacrine, rivastigmine, donepezil, and/or galantamine andother drugs and nutritive supplements, and, optionally, apharmaceutically acceptable carrier and/or a diluent and/or anexcipient.

In a specific embodiment, the invention provides a therapeuticcomposition comprising an antibody as described herein, particularly amonoclonal antibody, including any functionally equivalent antibody orfunctional parts thereof, further comprising at least one cholinesteraseinhibitor (ChEIs).

In another specific embodiment, the invention provides a therapeuticcomposition comprising an antibody as described herein, particularly amonoclonal antibody, including any functionally equivalent antibody orfunctional parts thereof, further comprising at least one additionalcompound selected from the group consisting of tacrine, rivastigmine,donepezil, galantamine, niacin and memantine.

In still another embodiment of the invention therapeutic compositionsare provided comprising an antibody as described herein, particularly amonoclonal antibody, including any functionally equivalent antibody orfunctional parts thereof, further comprising at least one “atypicalantipsychotic” such as, for example clozapine, ziprasidone, risperidone,aripiprazole or olanzapine for the treatment of positive and negativepsychotic symptoms including hallucinations, delusions, thoughtdisorders (manifested by marked incoherence, derailment, tangentiality),and bizarre or disorganized behavior, as well as anhedonia, flattenedaffect, apathy, and social withdrawal, and, optionally, furthercomprising a pharmaceutically acceptable carrier and/or a diluent and/oran excipient.

Other compounds that can be suitably used in mixtures in combinationwith an antibody according to the present invention, including anyfunctionally equivalent antibody or functional parts thereof, are, forexample, described in WO 2004/058258 (see especially pages 16 and 17)including therapeutic drug targets (page 36-39), alkanesulfonic acidsand alkanolsulfuric acids (pages 39-51), cholinesterase inhibitors(pages 51-56), NMDA receptor antagonists (pages 56-58), estrogens (pages58-59), non-steroidal anti-inflammatory drugs (pages 60-61),antioxidants (pages 61-62), peroxisome proliferators-activated receptor(PPAR) agonists (pages 63-67), cholesterol-lowering agents (pages68-75); amyloid inhibitors (pages 75-77), amyloid formation inhibitors(pages 77-78), metal chelators (pages 78-79), anti-psychotics andanti-depressants (pages 80-82), nutritional supplements (pages 83-89)and compounds increasing the availability of biologically activesubstances in the brain (see pages 89-93) and prodrugs (pages 93 and94), which document is incorporated herein by reference.

In particular, the therapeutic composition according to the inventioncomprises the monoclonal antibody and/or the biologically activesubstance, including any functionally equivalent antibody or functionalparts thereof, in a therapeutically effective amount.

In one embodiment, the invention relates to a method of producing anantibody as described herein, particularly a monoclonal antibodyincluding any functionally equivalent antibody or functional partsthereof, which method comprises raising in a suitable host organism anantibody against a supramolecular antigenic construct comprising anantigenic peptide corresponding to the amino acid sequence of theβ-amyloid peptide or a fragment thereof, particularly of β-amyloidpeptide Aβ₂₂₋₃₅ and Aβ₂₉₋₄₀, respectively, modified with hydrophilicmoieties, particularly a polyethylene glycol (PEG) moiety, wherein saidhydrophilic moiety is covalently bound to each terminus through an aminoacid such as, for example, lysine or any other suitable amino acid oramino acid analogue capable of serving as a linker molecule; andisolating the antibody.

In one embodiment, the invention relates to the use of an antibodyaccording to the invention and as described herein, particularly amonoclonal antibody including any functionally equivalent antibody orfunctional parts thereof and/or of a pharmaceutical compositionaccording to the invention and as described herein, or of a therapeuticcomposition according to the invention and as described herein for thepreparation of a medicament for treating or alleviating the effects ofdiseases and disorders in a subject, which are caused by or associatedwith amyloid or amyloid-like proteins including, but not limited to,amyloidosis, endocrine tumors, and macular degeneration.

In one embodiment, the invention relates to a method for the preparationof a pharmaceutical or therapeutic composition according to theinvention and as described herein using an antibody according to theinvention and as described herein, particularly a monoclonal antibodyincluding any functionally equivalent antibody or functional partsthereof for use in treating or alleviating the effects of diseases anddisorders in a subject, which are caused by or associated with amyloidor amyloid-like proteins including, but not limited to, amyloidosis,endocrine tumors, and macular degeneration.

In one embodiment, the invention provides a method for the preparationof a medicament using an antibody, particularly a monoclonal antibodyincluding any functionally equivalent antibody or functional partsthereof, a pharmaceutical or therapeutic composition according to theinvention and as described herein, for preventing, treating oralleviating the effects of diseases and disorders in a subject, whichare caused by or associated with amyloid or amyloid-like proteinsincluding, but not limited to, amyloidosis, endocrine tumors, andmacular degeneration.

In a specific embodiment, the invention relates to a method for thepreparation of a pharmaceutical composition using particularly anantibody according to the invention or as described herein, particularlya monoclonal antibody including any functionally equivalent antibody orfunctional parts thereof, comprising formulating said antibody in apharmaceutically acceptable form, particularly such that the antibody iscomprised in the composition in a therapeutically effective amount.

In one aspect of the invention, a method is provided for reducing theplaque load in the brain of a subject, particularly a mammal, butespecially a human suffering from a disease or condition associated withan increased plaque load in the brain comprising administering to asubject in need thereof, particularly a mammal, more particularly ahuman in need of such a treatment, a therapeutically effective amount ofan antibody, particularly a monoclonal antibody including anyfunctionally equivalent antibody or functional parts thereof, or acomposition or a mixture according to the invention and as describedherein. In a further aspect of the invention the monoclonal antibodiesused in such methods are ACI-11-Ab-9 having the polypeptide sequencesSEQ ID NOs: 7-8 or ACI-12-Ab-11 having the polypeptide sequences SEQ IDNO: 9-10, or a functional part thereof as described herein. Inparticular, the monoclonal antibody is produced by the hybridomaFG1F9E4, deposited on May 25, 2007 as DSM ACC2845, or the hybridomaFK2A6A6, deposited on May 25, 2007 as DSM ACC2846. In particular, theplaque load is reduced by at least 20%, particularly by at least 25%,more particularly by at least 30%, even more particularly by more than30%.

In another aspect of the invention, a method is provided for reducingthe amount of plaques in the brain of a subject, particularly a mammal,but especially a human suffering from a disease or condition associatedwith an increased plaque load in the brain comprising administering to asubject in need thereof, particularly a mammal, more particularly ahuman in need of such a treatment, a therapeutically effective amount ofan antibody, particularly a monoclonal antibody including anyfunctionally equivalent antibody or functional parts thereof, or acomposition or a mixture according to the invention and as describedherein. In a further aspect of the invention the monoclonal antibodiesused in such methods are ACI-11-Ab-9 having the polypeptide sequencesSEQ ID NOs: 7-8 or ACI-12-Ab-11 having the polypeptide sequences SEQ IDNO: 9-10, or a functional part thereof as described herein. Inparticular, the monoclonal antibody is produced by the hybridomaFG1F9E4, deposited on May 25, 2007 as DSM ACC2845, or the hybridomaFK2A6A6, deposited on May 25, 2007 as DSM ACC2846. In particular, theamount of plaques in the brain is reduced by at least 10%, particularlyby at least 15%, more particularly by more than 15%.

In still another aspect of the invention, a method is provided fordecreasing the total amount of soluble Aβ in the brain of a subject,particularly a mammal, but especially a human suffering from a diseaseor condition associated with increased concentrations of soluble Aβ inthe brain comprising administering to a subject in need thereof,particularly a mammal, more particularly a human in need of such atreatment, a therapeutically effective amount of an antibody,particularly a monoclonal antibody including any functionally equivalentantibody or functional parts thereof, or a composition or a mixtureaccording to the invention and as described herein. In a further aspectof the invention the monoclonal antibodies used in such methods areACI-11-Ab-9 having the polypeptide sequences SEQ ID NOs: 7-8 orACI-12-Ab-11 having the polypeptide sequences SEQ ID NO: 9-10, or afunctional part thereof as described herein. In particular, themonoclonal antibody is produced by the hybridoma FG1F9E4, deposited onMay 25, 2007 as DSM ACC2845, or the hybridoma FK2A6A6, deposited on May25, 2007 as DSM ACC2846.

In still another aspect of the invention, a method is provided forpreventing, treating or alleviating the effects of diseases anddisorders caused by or associated with amyloid or amyloid-like proteinsincluding, but not limited to, amyloidosis, endocrine tumors, andmacular degeneration, in a subject, particularly a mammal or a humanaffected by such a disorder, by administering a therapeuticallyeffective amount of an antibody, particularly a monoclonal antibody,including any functionally equivalent antibody or functional partsthereof, or a composition or a mixture according to the invention and asdescribed herein to a subject, particularly a mammal, more particularlya human in need of such a treatment.

In still another aspect of the invention, a method is provided forretaining or increasing cognitive memory capacity in a subject,particularly a mammal exhibiting an amyloid-associated disease orcondition comprising administering to a subject, particularly a mammal,more particularly a human in need of such a treatment, a therapeuticallyeffective amount of an antibody, particularly a monoclonal antibodyincluding any functionally equivalent antibody or functional partsthereof, or a composition or a mixture according to the invention or asdescribed herein.

In one embodiment, the invention relates to a hybridoma cell linecharacterized in that it produces a monoclonal antibody according to theinvention or as described herein.

In particular, the invention relates to a hybridoma cell linecharacterized in that it produces a monoclonal antibody, which antibodyhas the characteristic properties of an antibody produced by hybridomaFG1F9E4, deposited on May 25, 2007 and given the DSMZ deposit accessionnumber DSM ACC2845.

In a specific embodiment of the invention, hybridoma cell line FG1F9E4,deposited on May 25, 2007 and given the DSMZ deposit accession numberDSM ACC2845 is provided.

In particular, the invention relates to a hybridoma cell linecharacterized in that it produces a monoclonal antibody which antibodyhas the characteristic properties of an antibody produced by hybridomaFK2A6A6, deposited on May 25, 2007 and given the DSMZ deposit accessionnumber DSM ACC2846.

In a specific embodiment of the invention, hybridoma cell line FK2A6A6,deposited on May 25, 2007 and given the DSMZ deposit accession numberDSM ACC2846 is provided.

In one embodiment, the invention relates to a method of diagnosis of anamyloid-associated disease or condition in a patient comprisingdetecting the immunospecific binding of an antibody as described herein,particularly a monoclonal antibody including any functionally equivalentantibody or functional parts thereof, to an epitope of the amyloidprotein in a sample or in situ which includes the steps of

(a) bringing the sample or a specific body part or body area suspectedto contain the amyloid protein into contact with an antibody accordingto the invention, which antibody binds an conformational epitope of theamyloid protein;

(b) allowing the antibody to bind to the amyloid protein to form animmunological complex;

(c) detecting the formation of the immunological complex, particularlysuch that presence or absence of the immunological complex correlateswith presence or absence of amyloid protein; and

(d) correlating the presence or absence of the immunological complexwith the presence or absence of amyloid protein in the sample orspecific body part or area.

In a specific embodiment the composition of step (a) comprises acombination of antibodies for the treatment of said patient

In one embodiment, a method of determining the extent of amyloidogenicplaque burden in a tissue of a subject is provided comprising

obtaining a sample representative of the tissue under investigation;

testing said sample for the presence of amyloid plaque with an antibodyaccording to the invention and as described herein, particularly amonoclonal antibody including any functionally equivalent antibody orfunctional parts thereof;

determining the amount of antibody bound to the sample, particularlysuch that presence or absence of the immunological complex correlateswith presence or absence of amyloid plaque; and

calculating the plaque burden in the tissue.

In one embodiment, a method for diagnosing a predisposition to anamyloid-associated disease or condition in a patient is providedcomprising detecting the specific binding of an antibody as describedherein, particularly a monoclonal antibody including any functionallyequivalent antibody or functional parts thereof, to an epitope of theamyloid protein in a sample or in situ which includes the steps of

(a) bringing the sample or a specific body part or body area suspectedto contain the amyloid protein into contact with the antibody, whereinthe antibody binds an conformational epitope of the amyloid protein;

(b) allowing the antibody to bind to any amyloid protein in the sampleto form an immunological complex;

(c) detecting the formation of the immunological complex; and

(d) correlating the presence or absence of the immunological complexwith the presence or absence of amyloid protein in the sample orspecific body part or area,

(e) comparing the amount of said immunological complex to a normalcontrol value,

wherein an increase in the amount of said complex compared to a normalcontrol value indicates that said patient is suffering from or is atrisk of developing an amyloid-associated disease or condition.

In one embodiment, a method is provided for monitoring minimal residualdisease in a patient following treatment with an antibody or acomposition according to the invention, wherein said method comprises:

(a) bringing the sample or a specific body part or body area suspectedto contain the amyloid protein into contact with an antibody accordingto the invention and as described herein, particularly a monoclonalantibody including any functionally equivalent antibody or functionalparts thereof, which antibody binds an conformational epitope of theamyloid protein;

(b) allowing the antibody to bind to the amyloid protein to form animmunological complex;

(c) detecting the formation of the immunological complex; and

(d) correlating the presence or absence of the immunological complexwith the presence or absence of amyloid protein in the sample orspecific body part or area,

(e) comparing the amount of said immunological complex to a normalcontrol value,

wherein an increase in the amount of said complex compared to a normalcontrol value indicates that said patient still suffers from a minimalresidual disease.

In a specific embodiment the composition of step (a) comprises acombination of antibodies for the treatment of said patient.

In one embodiment, a method is provided for predicting responsiveness ofa patient being treated with an antibody or a composition according tothe invention comprising

(a) bringing the sample or a specific body part or body area suspectedto contain the amyloid protein into contact with an antibody accordingto the invention and as described herein, particularly a monoclonalantibody including any functionally equivalent antibody or functionalparts thereof, which antibody binds an conformational epitope of theamyloid protein;

(b) allowing the antibody to bind to the amyloid protein to form animmunological complex;

(c) detecting the formation of the immunological complex; and

(d) correlating the presence or absence of the immunological complexwith the presence or absence of amyloid protein in the sample orspecific body part or area, and

(e) comparing the amount of said immunological complex before and afteronset of the treatment,

wherein an decrease in the amount of said immunological complexindicates that said patient has a high potential of being responsive tothe treatment.

In a specific embodiment the composition of step (a) comprises acombination of antibodies for the treatment of said patient

In one embodiment, the invention relates to a test kit for the detectionand diagnosis of amyloid-associated diseases and conditions comprisingan antibody according to the invention and as described herein,particularly a monoclonal antibody including any functionally equivalentantibody or functional parts thereof and instructions for using theantibody for the purpose of binding to amyloid protein to form animmunological complex and detecting the formation of the immunologicalcomplex such that presence or absence of the immunological complexcorrelates with presence or absence of amyloid protein.

These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiment and the appended claims.

BRIEF DESCRIPTION OF FIGURES AND SEQUENCES

FIG. 1 shows the results of an epitope mapping study of the murinemonoclonal antibody ACI-11-Ab-9 performed by ELISA using a peptidelibrary of overlapping peptides covering the complete amino acidsequence of Aβ 1-42. Binding to the complete Aβ 1-42 was used aspositive control. All other peptides were 8-10 aa long. The peptidenumber corresponds to the aa in the Aβ 1-42 sequence on which thepeptide starts. Results are expressed as O.D.

FIG. 2 shows the results of an epitope mapping study of the murinemonoclonal antibody ACI-11-Ab-9 performed by ELISA using longer peptidecovering Aβ 1-28, 17-40, 1-40, 1-42A (Anaspec), or 1-42B (Bachem).Results are expressed as O.D., after subtraction of the background.Results show the mean±1 standard error of 2 independent experiments.

FIG. 3 shows the results of an epitope mapping study of the murinemonoclonal antibody ACI-12-Ab-11 performed by ELISA using a peptidelibrary of overlapping peptides covering the complete amino acidsequence of Aβ1-42. Binding to the complete Aβ 1-42 was used as positivecontrol. All other peptides were 8-10 aa long. The peptide numbercorresponds to the aa in the Aβ 1-42 sequence on which the peptidestarts. Results are expressed as O.D.

FIG. 4 shows the results of an epitope mapping study of the murinemonoclonal antibody ACI-11-Ab-9 performed by ELISA using longer peptidecovering. Aβ 1-28, 17-40, 1-40, 1-42A (Anaspec), or 1-42B (Bachem).Results are expressed as O.D., after subtraction of the background.Results show the mean±1 standard error of 2 independent experiments.

FIG. 5 depicts the ACI-11-Ab-9-mediated inhibition of Aβ1-42 aggregationat a 1:100 and 1:10 antibody to Aβ1-42 molar ratio. Results show mean±1standard error of 2 independent experiments.

FIG. 6 depicts the ACI-11-Ab-9-mediated disaggregation of pre-aggregatedAβ1-42 at a 1:100 and 1:10 antibody to Aβ1-42 molar ratio. Results showmean±1 standard error of 3 independent experiments.

FIG. 7 depicts the ACI-12-Ab-11-mediated inhibition of Aβ1-42aggregation at a 1:100 and 1:10 antibody to Aβ1-42 molar ratio. Resultsshow mean±1 standard error of 2 independent experiments.

FIG. 8 depicts the ACI-12-Ab-11-mediated disaggregation ofpre-aggregated Aβ1-42 at a 1:100 and 1:10 antibody to Aβ1-42 molarratio. Results show mean±1 standard error of 2 independent experiments.

FIG. 9 depicts the binding of ACI-12-Ab-11 antibody (A) and controlantibody 6E10 (B) to monomers and oligomers of the Aβ1-42 peptide. Theresults are reported as mean (±SEM) optical density (O.D.) values ofthree independent experiments.

FIG. 10 schematically depicts steps in the ELISA assay that can be usedto analyze the binding of rhApoE4 to Aβ₄₂-biotin.

FIG. 11 represents the results obtained from development of an ELISAassay for rhApoE4 to Aβ₄₂-biotin binding. To optimize the concentrationsof rhApoE4 and Aβ₄₂-biotin, dilutions of rhApoE4 are tested with aconstant concentration of Aβ₄₂-biotin.

FIG. 12 depicts the effect of excess of Aβ₄₂-biotin on the binding ofAβ₄₂-biotin complexed to rhApoE4 in the described ELISA assay.

FIG. 13 depicts a sample determination of the optimal concentration ofAβ₄₂-biotin for the described ELISA assay.

Table 1 sets forth the antibodies and antigenic constructs used forraising certain antibodies described herein.

Table 2. Binding of Aβ peptides to ACI-11-Ab-9 and ACI-12-Ab-11. Resultsare expressed as O.D. after background subtraction.

Table 3. Binding of ACI-11-Ab-9 and ACI-12-Ab-11 to 33 overlappingpeptides of Aβ 1-42 as analyzed by ELISA. Binding to the complete Aβ1-42 was used as positive control. All other peptides were 8-10 aa long.The peptide number corresponds to the aa in the Aβ 1-42 sequence onwhich the peptide starts. Results are expressed as O.D.

Table 4 Binding of the ACI-12-Ab-11 (clone FK2A6A6) antibody to monomersand oligomers of the Aβ1-42 peptide. Results are expressed as O.D.values.

Table 5 Binding of the 6E10 control antibody antibody to monomers andoligomers of the Aβ1-42 peptide. Results are expressed as O.D. values.

SEQ ID NO: 1: Antigenic peptide Aβ₂₂₋₃₅

SEQ ID NO: 2: Antigenic peptide Aβ₂₉₋₄₀

SEQ ID NO: 3: AB-peptide fragment Aβ₁₋₂₈

SEQ ID NO: 4: AB-peptide fragment Aβ₁₇₋₄₀

SEQ ID NO: 5 AB-peptide fragment Aβ₁₋₄₀

SEQ ID NO 6: AB-peptide fragment Aβ₁₋₄₂

SEQ ID NO: 7 Amino Acid sequence of the light chain variable domainsequence of ACI-11-Ab-9.

SEQ ID NO: 8 Amino Acid sequence of the heavy chain variable domainsequence of ACI-11-Ab-9.

SEQ ID NO: 9 Amino Acid sequence of the light chain variable domainsequence of ACI-12-Ab-11.

SEQ ID NO: 10 Amino Acid sequence of the heavy chain variable domainsequence of ACI-12-Ab-11.

SEQ ID NO: 11 Amino Acid sequence of the light chain CDR1

SEQ ID NO: 12 Amino Acid sequence of the light chain CDR2

SEQ ID NO: 13 Amino Acid sequence of the light chain CDR3

SEQ ID NO: 14 Amino Acid sequence of the heavy chain CDR1

SEQ ID NO: 15 Amino Acid sequence of the heavy chain CDR2

SEQ ID NO: 16 Amino Acid sequence of the heavy chain CDR3

SEQ ID NO: 17 Polynucleotide sequence encoding the light chain variabledomain sequence of ACI-11-Ab-9.

SEQ ID NO: 18 Polynucleotide sequence encoding the heavy chain variabledomain sequence of ACI-11-Ab-9.

SEQ ID NO: 19 Polynucleotide sequence encoding the light chain variabledomain sequence of ACI-12-Ab-11.

SEQ ID NO: 20 Polynucleotide sequence encoding the light chain variabledomain sequence of ACI-12-Ab-11.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The terms “polypeptide”, “peptide”, and “protein”, as used herein, areinterchangeable and are defined to mean a biomolecule composed of aminoacids linked by a peptide bond.

The terms “a”, “an” and “the” as used herein are defined to mean “one ormore” and include the plural unless the context is inappropriate.

The terms “detecting” or “detected” as used herein mean using knowntechniques for detection of biologic molecules such as immunochemical orhistological methods and refer to qualitatively or quantitativelydetermining the presence or concentration of the biomolecule underinvestigation.

“Amyloid β, Aβ or β-amyloid” is an art recognized term and refers toamyloid β proteins and peptides, amyloid β precursor protein (APP), aswell as modifications, fragments and any functional equivalents thereof.As used herein, amyloid β refers to any fragment produced by proteolyticcleavage of APP but especially those fragments which are involved in orassociated with the amyloid pathologies including, but not limited to,Aβ₁₋₃₈, Aβ₁₋₃₉, Aβ₁₋₄₀, Aβ₁₋₄₁ Aβ₁₋₄₂ and Aβ₁₋₄₃.

The structure and sequences of the amyloid β peptides as mentioned aboveare well known to those of ordinary skill in the art and methods ofproducing said peptides or of extracting them from brain and othertissues are described, for example, in Glenner and Wong, Biochem BiophysRes Comm 129, 885-890 (1984). Moreover, amyloid β peptides are alsocommercially available in various forms.

“Aβ Fibril” or “Aβ Filament” or “amyloid fibrils” are polymeric forms ofmonomeric protein forming individual or bundled fibers with constantfiber diameter which are insoluble in aqueous medium and contain largeamounts of a cross-β structure in their core; mostly with beta-strandsperpendicular to the fibril axis.

“Monomeric Aβ” or “Aβ monomer” are completely solubilized amyloid 0protein without aggregated complexes in aqueous medium.

“Proto-fibrils” or “proto-fibrillar preparation” as used herein refersto high molecular weight fractions of polymeric Aβ amyloid peptides,which are enriched with soluble amyloid Aβ oligomers.

“Polymeric soluble amyloid” and “oligomeric amyloid peptides Aβ” and “Aβoligomer” are used interchangeably herein and refer to multipleaggregated monomers of amyloid peptides, or of amyloid-like peptides, orof modified or truncated amyloid peptides or of other derivates ofamyloid peptides forming oligomeric or polymeric structures which aresoluble both in vitro in aqueous medium and in vivo in the mammalian orhuman body more particularly in the brain, but particularly to multipleaggregated monomers of amyloid peptides or of modified or truncatedamyloid peptides or of derivatives thereof, which are soluble in themammalian or human body more particularly in the brain.

“Polymeric soluble amyloid Aβ peptides” and “oligomeric amyloid Aβpeptides” and “Aβ oligomer” are used interchangeably herein and refersto multiple aggregated monomers of amyloid Aβ peptides, or of modifiedor truncated amyloid Aβ peptides or of other derivates of amyloid Aβpeptides forming oligomeric or polymeric structures which are solubleboth in vitro in aqueous medium and in vivo in the mammalian or humanbody more particularly in the brain, but particularly to multipleaggregated monomers of amyloid β (Aβ) or of modified or truncatedamyloid β (Aβ) peptides or of derivatives thereof, which are soluble inthe mammalian or human body more particularly in the brain.

With respect to a monoclonal antibody including any functionallyequivalent antibody or functional parts thereof, which antibody binds toAβ monomeric peptide 1-40 particularly to Aβ monomeric peptide 1-40 andto soluble polymeric and/or oligomeric amyloid peptide comprising aplurality of Aβ1-42 monomeric peptides but shows a substantially weakerbinding to Aβ monomeric peptide 1-28 and an intermediated binding tomonomeric peptide 1-42 and essentially no binding to Aβ monomericpeptide 17-40, by a “substantially weaker binding” a binding is meant,which is at least about 80%, particularly at least about 85%, moreparticularly at least about 90% but especially at least about 95% lessthan the binding to Aβ monomeric peptide 1-40.

With respect to a monoclonal antibody including any functionallyequivalent antibody or functional parts thereof, which antibody binds toAβ monomeric peptide 1-42 and polymeric soluble Aβ peptides comprising aplurality of Aβ₁₋₄₂ monomeric peptides and Aβ fibrils or fibersincorporating a plurality of said polymeric peptides, but shows asubstantially weaker binding to Aβ monomeric peptide 1-28 andessentially no binding to Aβ monomeric peptide 17-40, by a“substantially weaker binding” a binding is meant, which is at leastabout 60%, particularly at least about 65%, more particularly at leastabout 70%, even more particularly at least about 80%, but especially atleast about 90% and up to 100% less than the binding to Aβ monomericpeptide 1-42.

With respect to a monoclonal antibody including any functionallyequivalent antibody or functional parts thereof, which antibody binds toAβ monomeric peptide 1-40 particularly to Aβ monomeric peptide 1-40 andto soluble polymeric and/or oligomeric amyloid peptide comprising aplurality of Aβ1-42 monomeric peptides but shows a substantially weakerbinding to Aβ monomeric peptide 1-28 and an intermediated binding tomonomeric peptide 1-42 and essentially no binding to Aβ monomericpeptide 17-40, by an “intermediate binding” a binding is meant, which isat least about 60%, particularly at least about 65%, more particularlyat least about 70%, even more particularly at least about 80%, less thanthe binding to Aβ monomeric peptide 1-40.

With respect to a monoclonal antibody including any functionallyequivalent antibody or functional parts thereof, which antibody binds toAβ monomeric peptide 1-40 particularly to Aβ monomeric peptide 1-40 andto soluble polymeric and/or oligomeric amyloid peptide comprising aplurality of Aβ1-42 monomeric peptides but shows a substantially weakerbinding to Aβ monomeric peptide 1-28 and an intermediated binding tomonomeric peptide 1-42 and essentially no binding to Aβ monomericpeptide 17-40, by “essentially no binding” a binding is meant, which isat least about 95%, particularly at least about 98%, but especially atleast about 99% and up to 100% less than the binding to Aβ monomericpeptide 1-40.

With respect to a monoclonal antibody including any functionallyequivalent antibody or functional parts thereof, which antibody binds toAβ monomeric peptide 1-42 and polymeric soluble Aβ peptides comprising aplurality of Aβ₁₋₄₂ monomeric peptides and Aβ fibrils or fibersincorporating a plurality of said polymeric peptides, but shows asubstantially weaker binding to Aβ monomeric peptide 1-28 andessentially no binding to Aβ monomeric peptide 17-40, by “essentially nobinding” a binding is meant, which is at least about 85%, particularlyat least about 90%, more particularly at least about 95%, even moreparticularly at least about 98%, but especially at least about 99% andup to 100% less than the binding to Aβ monomeric peptide 1-42.

The binding of the antibody according to the invention as describedherein, particularly a monoclonal antibody including any functionallyequivalent antibody or functional parts thereof, to Aβ monomericpeptides is determined by an ELISA-type assay, particularly by an ELISAassay using biotinylated Aβ monomeric peptides, but especially by anELISA assay as described in Example 16 below.

The language “diseases and disorders which are caused by or associatedwith amyloid or amyloid-like proteins” includes, but is not limited to,diseases and disorders caused by the presence or activity ofamyloid-like proteins in monomeric, fibril, or polymeric state, or anycombination of the three. Such diseases and disorders include, but arenot limited to, amyloidosis, endocrine tumors, and macular degeneration.

The term “optically associated amyloid diseases” refers todrusen-related optic neuropathy, cataract due to beta-amyloiddeposition, and macular degernation.

The term “amyloidosis” refers to a group of diseases and disordersassociated with amyloid plaque formation including, but not limited to,secondary amyloidosis and age-related amyloidosis such as diseasesincluding, but not limited to, neurological disorders such asAlzheimer's Disease (AD), including diseases or conditions characterizedby a loss of cognitive memory capacity such as, for example, mildcognitive impairment (MCI), Lewy body dementia, Down's syndrome,hereditary cerebral hemorrhage with amyloidosis (Dutch type); the GuamParkinson-Dementia complex; as well as other diseases which are based onor associated with amyloid-like proteins such as progressivesupranuclear palsy, multiple sclerosis; Creutzfeld Jacob disease,Parkinson's disease, HIV-related dementia, ALS (amyotropic lateralsclerosis), inclusion-body myositis (IBM), Adult Onset Diabetes; andsenile cardiac amyloidosis, and various eye diseases including maculardegeneration, drusen-related optic neuropathy, and cataract due tobeta-amyloid deposition

By “isolated” is meant a biological molecule free from at least some ofthe components with which it naturally occurs.

The terms “antibody” or “antibodies” as used herein are art recognizedterms and are understood to refer to molecules or active fragments ofmolecules that bind to known antigens, particularly to immunoglobulinmolecules and to immunologically active portions of immunoglobulinmolecules, i.e. molecules that contain a binding site thatimmunospecifically binds an antigen. The immunoglobulin according to theinvention can be of any type (IgG, IgM, IgD, IgE, IgA and IgY) or class(IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclasses of immunoglobulinmolecule.

“Antibodies” are intended within the scope of the present invention toinclude monoclonal, polyclonal, chimeric, single chain, bispecific orbi-effective, simianized, human and humanized antibodies as well asactive fragments thereof. Examples of active fragments of molecules thatbind to known antigens include Fab, F(ab′)₂, scFv and Fv fragments,including the products of an Fab immunoglobulin expression library andepitope-binding fragments of any of the antibodies and fragmentsmentioned above.

Such active fragments can be derived from an antibody of the presentinvention by a number of art-known techniques. For example, purifiedmonoclonal antibodies can be cleaved with an enzyme, such as pepsin, andsubjected to HPLC gel filtration. The appropriate fraction containingFab fragments can then be collected and concentrated by membranefiltration and the like. For further description of general techniquesfor the isolation of active fragments of antibodies, see for example,Khaw, B. A. et al. J. Nucl. Med. 23:1011-1019 (1982); Rousseaux et al.Methods Enzymology, 121:663-69, Academic Press, 1986.

A “humanized antibody” refers to a type of engineered antibody havingits CDRs derived from a non-human donor immunoglobulin, the remainingimmunoglobulin-derived parts of the molecule being derived from one (ormore) human immunoglobulin(s). In addition, framework support residuesmay be altered to preserve binding affinity. Methods to obtain“humanized antibodies” are well known to those skilled in the art. (see,e.g., Queen et al., Proc. Natl Acad Sci USA, 86:10029-10032 (1989),Hodgson et al., Bio/Technology, 9:421 (1991)).

A “humanized antibody” may also be obtained by a novel geneticengineering approach that enables production of affinity-maturedhumanlike polyclonal antibodies in large animals such as, for example,rabbits (see, e.g. U.S. Pat. No. 7,129,084).

The term “monoclonal antibody” is also well recognized in the art andrefers to an antibody that is mass produced in the laboratory from asingle clone and that recognizes only one antigen. Monoclonal antibodiesare typically made by fusing a normally short-lived, antibody-producingB cell to a fast-growing cell, such as a cancer cell (sometimes referredto as an “immortal” cell). The resulting hybrid cell, or hybridoma,multiplies rapidly, creating a clone that produces large quantities ofthe antibody. For the purpose of the present invention, “monoclonalantibody” is also to be understood to comprise antibodies that areproduced by a mother clone which has not yet reached full monoclonality.

The term “CDR” refers to the hypervariable region of an antibody. Theterm “hypervariable region”, “HVR”, or “HV”, when used herein refers tothe regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six hypervariable regions; three in the VH (H1, H2, H3), andthree in the VL (L1, L2, L3). A number of hypervariable regiondelineations are in use and are encompassed herein. The KabatComplementarity Determining Regions are based on sequence variabilityand are the most commonly used (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)).

The letters “HC” and “LC” preceding the term “CDR” refer, respectively,to a CDR of a heavy chain and a light chain. Chothia refers instead tothe location of the structural loops (Chothia and Lesk J. Mol. Biol.196:901-917 (1987)). The AbM hypervariable regions represent acompromise between the Kabat CDRs and Chothia structural loops, and areused by Oxford Molecular's AbM antibody modeling software. The “contact”hypervariable regions are based on an analysis of the available complexcrystal structures. The residues from each of these hypervariableregions are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

Hypervariable regions may comprise “extended hypervariable regions” asfollows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96(L3) in the VL and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102,or 95-102 (H3) in the VH. The variable domain residues are numberedaccording to Kabat et al., supra, for each of these definitions.

The term “variable domain residue numbering as in Kabat” or “amino acidposition numbering as in Kabat,” and variations thereof, refers to thenumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991).

Using this numbering system, the actual linear amino acid sequence maycontain fewer or additional amino acids corresponding to a shorteningof, or insertion into, a FR or HVR of the variable domain. For example,a heavy chain variable domain may include a single amino acid insert(residue 52a according to Kabat) after residue 52 of H2 and insertedresidues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat)after heavy chain FR residue 82. The Kabat numbering of residues may bedetermined for a given antibody by alignment at regions of homology ofthe sequence of the antibody with a “standard” Kabat numbered sequence.

“Functionally equivalent antibody” is understood within the scope of thepresent invention to refer to an antibody which substantially shares atleast one major functional property with an antibody, for examplefunctional properties herein described including, but not limited to:binding specificity to the β-amyloid protein, particularly to the Aβ₁₋₄₂protein, and more particularly to the 4-16 epitopic region of the Aβ₁₋₄₂protein, immunoreactivity in vitro, inhibition of aggregation of theAβ₁₋₄₂ monomers into high molecular polymeric fibrils and/ordisaggregation of preformed Aβ₁₋₄₂ polymeric fibrils, and/or a β-sheetbreaking property and alleviating the effects of diseases and disorderswhich are caused by or associated with amyloid or amyloid-like proteinsincluding, but not limited to, amyloidosis, endocrine tumors, andmacular degeneration, when administered prophylactically ortherapeutically. The antibodies can be of any class such as IgG, IgM, orIgA, etc or any subclass such as IgG1, IgG2a, etc and other subclassesdescribed herein above or known in the art, but particularly of the IgG4class. Further, the antibodies can be produced by any method, such asphage display, or produced in any organism or cell line, includingbacteria, insect, mammal or other type of cell or cell line whichproduces antibodies with desired characteristics, such as humanizedantibodies. Antibodies can also be formed by combining a Fab portion andan Fc region from different species.

The term “bispecific” or “bifunctional” and “bi-effective” is usedsynonymously within the scope of this application to characterize anantibody which exhibits both an inhibition property on amyloid oramyloid-like fiber formation as well as a disaggregation property ofamyloid or amyloid-like fibers.

The term “antigen” refers to an entity or fragment thereof which caninduce an immune response in an organism, particularly an animal, moreparticularly a mammal including a human. The term includes immunogensand regions thereof responsible for antigenicity or antigenicdeterminants.

As used herein, the term “soluble” means partially or completelydissolved in an aqueous solution.

Also as used herein, the term “immunogenic” refers to substances whichelicit or enhance the production of antibodies, T-cells or otherreactive immune cells directed against an immunogenic agent andcontribute to an immune response in humans or animals.

An immune response occurs when an individual produces sufficientantibodies, T-cells and other reactive immune cells against administeredimmunogenic compositions of the present invention to moderate oralleviate the disorder to be treated.

“Polymeric soluble amyloid” refers to multiple aggregated monomers ofamyloid peptides, or of amyloid-like peptides, or of modified ortruncated amyloid peptides or of other derivates of amyloid peptidesforming oligomeric or polymeric structures which are soluble in themammalian or human body more particularly in the brain, but particularlyrefer to multiple aggregated monomers of amyloid β (Aβ) or of modifiedor truncated amyloid β (Aβ) peptides or of derivatives thereof, whichare soluble in the mammalian or human body more particularly in thebrain.

The term “hybridoma” is art recognized and is understood by those ofordinary skill in the art to refer to a cell produced by the fusion ofan antibody-producing cell and an immortal cell, e.g. a multiple myelomacell. Such a hybrid cell is capable of producing a continuous supply ofantibody. See the definition of “monoclonal antibody” above and theExamples below for a more detailed description of one art known methodof fusion.

The term “carrier” as used herein means a structure in which antigenicpeptide or supramolecular construct can be incorporated into or can beassociated with, thereby presenting or exposing antigenic peptides orpart of the peptide to the immune system of a human or animal. Anyparticle that can be suitably used in animal or human therapy such as,for example, a vesicle, a particle or a particulate body may be used asa carrier within the context of the present invention. The term furthercomprises methods of delivery wherein supramolecular antigenic constructcompositions comprising the antigenic peptide may be transported todesired sites by delivery mechanisms. One example of such a deliverysystem utilizes colloidal metals such as colloidal gold. The term“carrier” further comprises delivery mechanisms known to those ofordinary skill in the art including, but not limited to, keyhole limpethemocyanin (KLH), bovine serum albumin (BSA) and other adjuvants.

In a supramolecular antigenic construct according to the presentinvention, the liposome may have a dual function in that it can be usedas a carrier comprising a supramolecular construct as described hereinand, at the same time, function as an adjuvant to increase or stimulatethe immune response within the target animal or human to be treated withthe therapeutic vaccine according to the invention. It is also to beunderstood that the supramolecular antigenic construct compositions ofthe present invention can further comprise additional adjuvants such as,for example, lipid A, alum, calcium phosphate, interleukin 1, and/ormicrocapsules of polysaccharides and proteins, but particularly adetoxified lipid A, such as monophosphoryl or diphosphoryl lipid A, oralum, further preservatives, diluents, emulsifiers, stabilizers, andother components that are known and used in vaccines in the art.Moreover, any adjuvant system known in the art can be used in thecomposition of the present invention. Such adjuvants include, but arenot limited to, Freund's incomplete adjuvant, Freund's completeadjuvant, polydispersed β-(1,4) linked acetylated mannan (“Acemannan”),TITERMAX® (polyoxyethylene-polyoxypropylene copolymer adjuvants fromCytRx Corporation), modified lipid adjuvants from Chiron Corporation,saponin derivative adjuvants from Cambridge Biotech, killed Bordetellapertussis, the lipopolysaccharide (LPS) of gram-negative bacteria, largepolymeric anions such as dextran sulfate, and inorganic gels such asalum, aluminum hydroxide, or aluminum phosphate.

Carrier proteins that can be used in the supramolecular antigenicconstruct compositions of the present invention include, but are notlimited to, maltose binding protein “MBP”; bovine serum albumin “BSA”;keyhole lympet hemocyanin “KLH”; ovalbumin; flagellin; thyroglobulin;serum albumin of any species; gamma globulin of any species; syngeneiccells; syngeneic cells bearing Ia antigens; and polymers of D- and/orL-amino acids.

Further, the term “therapeutically effective amount” refers to theamount of antibody which, when administered to a human or animal,elicits an immune response which is sufficient to result in atherapeutic effect in said human or animal. The effective amount isreadily determined by one of ordinary skill in the art following routineprocedures.

“Homology” between two sequences is determined by sequence identity. Iftwo sequences which are to be compared with each other differ in length,sequence identity preferably relates to the percentage of the nucleotideresidues of the shorter sequence which are identical with the nucleotideresidues of the longer sequence. Sequence identity can be determinedconventionally with the use of computer programs such as the Bestfitprogram (Wisconsin Sequence Analysis Package, Version 8 for Unix,Genetics Computer Group, University Research Park, 575 Science DriveMadison, Wis. 53711). Bestfit utilizes the local homology algorithm ofSmith and Waterman, Advances in Applied Mathematics 2 (1981), 482-489,in order to find the segment having the highest sequence identitybetween two sequences. When using Bestfit or another sequence alignmentprogram to determine whether a particular sequence has for example 95%identity with a reference sequence of the present invention, theparameters are preferably adjusted so that the percentage of identity iscalculated over the entire length of the reference sequence and homologygaps of up to 5% of the total number of the nucleotides in the referencesequence are permitted. When using Bestfit, the so-called optionalparameters are preferably left at their preset (“default”) values. Thedeviations appearing in the comparison between a given sequence and theabove-described sequences of the invention may be caused for instance byaddition, deletion, substitution, insertion or recombination. Such asequence comparison can preferably also be carried out with the program“fasta20u66” (version 2.0u66, September 1998 by William R. Pearson andthe University of Virginia; see also W. R. Pearson (1990), Methods inEnzymology 183, 63-98, appended examples andhttp://workbench.sdsc.edu/). For this purpose, the “default” parametersettings may be used.

As used herein a “conservative change” refers to alterations that aresubstantially conformationally or antigenically neutral, producingminimal changes in the tertiary structure of the mutant polypeptides, orproducing minimal changes in the antigenic determinants of the mutantpolypeptides, respectively, as compared to the native protein. Whenreferring to the antibodies and antibody fragments of the invention, aconservative change means an amino acid substitution that does notrender the antibody incapable of binding to the subject receptor. One ofordinary skill in the art will be able to predict which amino acidsubstitutions can be made while maintaining a high probability of beingconformationally and antigenically neutral. Such guidance is provided,for example in Berzofsky, (1985) Science 229:932-940 and Bowie et al.(1990) Science 247:1306-1310. Factors to be considered that affect theprobability of maintaining conformational and antigenic neutralityinclude, but are not limited to: (a) substitution of hydrophobic aminoacids is less likely to affect antigenicity because hydrophobic residuesare more likely to be located in a protein's interior; (b) substitutionof physiochemically similar, amino acids is less likely to affectconformation because the substituted amino acid structurally mimics thenative amino acid; and (c) alteration of evolutionarily conservedsequences is likely to adversely affect conformation as suchconservation suggests that the amino acid sequences may have functionalimportance. One of ordinary skill in the art will be able to assessalterations in protein conformation using well-known assays, such as,but not limited to microcomplement fixation methods (see, e.g. Wassermanet al. (1961) J. Immunol. 87:290-295; Levine et al. (1967) Meth.Enzymol. 11:928-936) and through binding studies usingconformation-dependent monoclonal antibodies (see, e.g. Lewis et al.(1983) Biochem. 22:948-954).

The term “hybridize” as used herein refers to conventional hybridizationconditions, preferably to hybridization conditions at which 5×SSPE, 1%SDS, 1×Denhardts solution is used as a solution and/or hybridizationtemperatures are between 35° C. and 70° C., preferably 65° C. Afterhybridization, washing is preferably carried out first with 2×SSC, 1%SDS and subsequently with 0.2×SSC at temperatures between 35° C. and 70°C., preferably at 65° C. (regarding the definition of SSPE, SSC andDenhardts solution see Sambrook et al. Molecular Biology: A LaboratoryManual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989).Stringent hybridization conditions as for instance described in Sambrooket al, supra, are particularly preferred. Particularly preferredstringent hybridization conditions are for instance present ifhybridization and washing occur at 65° C. as indicated above.Non-stringent hybridization conditions, for instance with hybridizationand washing carried out at 45° C. are less preferred and at 35° C. evenless.

The present invention may be understood more readily by reference to thefollowing detailed description of specific embodiments included herein.Although the present invention has been described with reference tospecific details of certain embodiments thereof, it is not intended thatsuch details should be regarded as limitations upon the scope of theinvention.

The present invention provides antibodies and functional parts thereofwhich are conformationally sensitive antibodies. These antibodiesrecognize specific epitopes on a wide variety of amyloid proteinicantigens. The antibodies are useful for diagnostic and therapeuticintervention in diseases and disorders which are caused by or associatedwith amyloid or amyloid-like proteins, and especially in Alzheimer'sDisease.

Antibodies may be administered to individuals to passively immunize themagainst a variety of diseases or disorders which are caused by orassociated with amyloid or amyloid-like proteins, such as Alzheimer'sdisease.

The antibodies provided herein are monoclonal or polyclonal antibodieshaving binding specificity for antigenic peptides involved in theinitiation, progression, and/or worsening of various diseases ordisorders which are caused by or associated with amyloid or amyloid-likeproteins, such as, Alzheimer's disease.

The antibodies according to the invention are prepared by immunizing ananimal, such as a mouse, rat, rabbit or any other animal species whichcan produce native or human antibodies, with a supramolecular antigenicconstruct composition.

The supramolecular antigenic constructs as disclosed herein generallycomprise peptides modified to enhance antigenic effect wherein suchpeptides are modified via pegylation (using polyethylene glycol ormodified polyethylene glycol), or modified via other methods such as bypalmitic acid, poly-amino acids (e.g. poly-glycine, poly-histidine),poly-saccharides (e.g. polygalacturonic acid, polylactic acid,polyglycolide, chitin, chitosan), synthetic polymers (polyamides,polyurethanes, polyesters) or co-polymers (e.g. poly(methacrylic acid)and N-(2-hydroxy) propyl methacrylamide) and the like.

Modification by palmitic acid (palmitoylation), while providing ananchor for the peptide in the liposome bilayer, due to the relativereduced length of the C_(16:0) fatty acid moiety leads to the peptidepractically laying on the liposome surface. Therefore, the cellsprocessing the antigen will have to take up the entire liposome with thepeptide, which, in the majority of cases, results in a slower immuneresponse in relative terms.

In one embodiment of the invention, a modified amyloid Aβ₂₂₋₃₅ peptideand Aβ₂₉₋₄₀, peptide, respectively, is used in the preparation of anantibody, particularly a monoclonal antibody according to the invention.The modified amyloid peptide may be synthesized following the methodreported in Nicolau et. al. 2002. The approach reported in Nicolau et alinvolves modifying the antigenic peptide by an on-resin grafting of alipophilic or hydrophobic moiety, to the terminal amino acid residues ofa pre-formed peptide resulting in a product of considerably high purity.In particular, a protected amino acid, particularly a Fmoc-protectedamino acid, is attached to a resin using known coupling chemistry. Theprotecting group is removed and a second protected amino acid residuecoupled. Standard automated peptide synthesis using known protectionchemistry, particularly Fmoc/tBu chemistry, and standard side-chainprotecting groups are then used to synthesize the Aβ antigenic peptideby coupling on amino acids 22 to 35 and 29-40, respectively, of amyloidprotein Aβ₁₋₄₂. to produce the peptide fragment. In a final step twofurther protected amino acids are coupled to the growing peptidefragment. The Mtt groups can then be selectively cleaved and coupled topalmitic acid. After washing of the resin, the protecting group isremoved and the resin simultaneously cleaved, followed by side-chaindeprotections using standard methodology. The final product can then beobtained in high purity and its identity confirmed by methods known inthe art such as, for example, electrospray mass spectrometry.

The lipophilic or hydrophobic moiety according to the present inventionmay be a fatty acid, a triglyceride or a phospholipid wherein the fattyacid carbon back bone has at least 10 carbon atoms. Particularly, thelipophilic or hydrophobic moiety is a fatty acids with a carbon backboneof at least approximately 14 carbon atoms and up to approximately 24carbon atoms, with each individual number of carbon atom falling withinthis range also being part of the present invention. More particularly,the lipophilic or hydrophobic moiety has a carbon backbone of at least14 carbon atoms. Examples of hydrophobic moieties include, but are notlimited to, palmitic acid, stearic acid, myristic acid, lauric acid,oleic acid, linoleic acid, linolenic acid and cholesterol or DSPE. In aspecific embodiment of the present invention the lipophilic orhydrophobic moiety is palmitic acid.

To enhance the immune response, another anchor/spacer can suitably beapplied to reconstitute the peptide in the liposome, e.g. polyethyleneglycol (PEG).

PEG is covalently attached to an amino acid residue bound at bothtermini of the peptide, in particular Glu, Cys or Lys amino acid residueor any other amino acid residue that can be suitably used to covalentlybind PEG to the peptide. At the other end of the chain a hydrophobicmoiety may be covalently bound to function as the anchoring element inthe liposome bilayer such as, for example, phosphatidyl ethanol amine(PEA). Thus, the liposome still functions as an adjuvant and the peptidebeing sufficiently far away from the bilayer can be processed alone andthus increases its immunogenicity as compared to the palmitoylatedantigen.

In certain embodiments, the supramolecular antigenic constructs usedwithin the scope of the present invention comprise a peptide sequence,covalently attached to pegylated lysine—one at each terminus. The lengthof the PEG (polyethylenglycol) chain may vary from n=8 to n=150.000 ormore, particularly from n=10 to n=80.000, more particularly from n=20 ton=10.000. In a specific embodiment of the invention the length of thePEG chain is not more than n=45, particularly between n=5 and n=40, moreparticularly between n=10 and n=30, and even more particularly n=10.

The supramolecular constructs described herein can be synthesized usingautomated peptide synthesis and known protection chemistry, particularlyFmoc/tBu chemistry and standard side-chain protecting groups. Typically,pegylation of peptides results in mixtures of regioisomers.

To achieve a site-specific attachment of a PEG-lipid conjugate to boththe C- and N-terminus of Aβ partially protected peptides may be used.For those peptide sequences containing internal Lys or His residues anorthogonally protected Lys(ivDde) is added to each terminus. Anadditional Gly may be added to the C-terminal end to facilitatesynthesis. The protecting group is removed and N-acetylated using aceticanhydride followed by selective cleavage of the ivDde groups.

A resin, particularly a 2-chlorotrityl resin, is to be favored which isacid sensitive and thus enables the isolation of protected peptides.

In a specific embodiment of the invention, the coupling reaction isperformed in the solution phase. Selective cleavage from the resin undermild conditions then release the internally protected peptides.

Solution-phase couplings were achieved successfully with the peptidesderived from a β-amyloid protein sequence such as, for example, aAβ₂₂₋₃₅ and Aβ₂₉₋₄₀, respectively, to a PEG molecule modified by a fattyacid-phosphatidylcholine such as, for example, DSPE. Separation of themono- and di-coupled products before final side-chain deprotections canbe achieved by using cation-exchange chromatography. Subsequent peptideside-chain deprotections leads to the isolation of the desiredconjugates with an acceptable purity. Purification can be achieved bymethods well known in the art such as, for example, HPLC. etc.

This approach to the synthesis of N- and C-terminal lipid-PEG β-amyloidantigens using protected peptides is applicable to a wide variety ofpeptide sequences.

Liposomal antigens according to the invention may then be prepared asdescribed in Nicolau et al., 2002. The modified amyloid Aβ antigenicpeptide, particularly the modified PEGylated Aβ₂₂₋₃₅ and Aβ₂₉₋₄₀antigenic peptide may be reconstituted in a construct consisting ofliposomes, particularly liposomes made of dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidyl ethanolamine (DMPEA),dimyristoyl phosphatidyl glycerol (DMPG) and cholesterol, optionallycontaining monophosphoryl lipid A.

In a specific embodiment of the invention liposomes with lipid A areused as adjuvant to prepare the anti-amyloid vaccine.Dimyristoylphosphatidyl-choline, -glycerol and cholesterol are mixed,particularly in a molar ratio of 0.9:1.0:0.7. A strong immunmodulatorsuch as, for example, monophosphoryl lipid A is then added at a suitableconcentration, particularly at a concentration of between 30 and 50 mgper mmol, more particularly at 40 mg per mmol of phospholipids. Themodified antigenic Aβ peptide is then added at a molar ratio peptide tophospholipids of between 1:30 and 1:200, particularly at a molar ratioof between 1:1000, 1:50, and 1:120, more particularly of 1:100. Solventsare removed, for example through evaporation, and the resulting filmhydrated with sterile buffer solution such as, for example PBS.

Liposomes may also be prepared by the crossflow injection technique asdescribed, for example, in Wagner et al (2002) Journal of LiposomeResearch Vol 12(3), pp 259-270. During the injection of lipid solutionsinto an aqueous buffer system, lipids tend to form “precipitates”,followed by self arrangement in vesicles. The obtained vesicle sizedepends on factors such as lipid concentration, stirring rate, injectionrate, and the choice of lipids. The preparation system may consist of acrossflow injection module, vessels for the polar phase (e.g. a PBSbuffer solution), an ethanol/lipid solution vessel and a pressuredevice, but particularly a nitrogen pressure device. While the aqueousor polar solution is pumped through the crossflow injection module theethanol/lipid solution is injected into the polar phase with varyingpressures applied.

The liposome still functions as an adjuvant and the peptide beingsufficiently far away from the bilayer can be processed alone and thusincreases its immunogenicity as compared to the palmitoylated antigen.

The free PEG terminus is covalently attached to a molecule ofphosphatidylethanolamine (where the fatty acid can be: myristic,palmitic, stearic, oleic etc. or a combination thereof) to function asthe anchoring element. This supramolecular structure may be anchored byreconstitution in liposomes consisting of phospholipids and cholesterol(phosphatidylethanol amine, phosphatidyl glycerol, cholesterol in variedmolar ratios. Other phospholipids can be used. Lipid A is used at aconcentration of approximately 40 μg/μmole of phospholipids.

In certain embodiments, the palmitoylated or pegylated supramolecularantigenic constructs comprise a peptide having the amino acid sequenceof β-amyloid. The peptides may also comprise or correspond to wholeamyloid beta peptide and active fragments thereof. Additionally,peptides useful for the present invention in particular comprise Aβ₂₂₋₃₅and Aβ₂₉₋₄₀, respectively, and active fragments thereof.

For eliciting and preparing antibodies and for determining immuogenicityof the modified Aβ antigenic construct a suitable animal selected fromthe group consisting of mice, rats, rabbits, pigs, birds, etc, butparticularly mice, especially C57BL/6 mice are immunized with theantigenic peptide. Immunogenicity of the antigenic construct isdetermined by probing sera samples in suitable time intervals afterimmunization using a immunoassay such as, for example, an ELISA assay.

The monoclonal antibodies of the present invention can be prepared usingclassical cloning and cell fusion techniques well known in the art. Theimmunogen (antigen) of interest, is typically administered (e.g.intraperitoneal injection) to wild type or inbred mice (e.g. BALB/c orespecially C57BL/6 mice), rats, rabbits or other animal species ortransgenic mice which can produce native or human antibodies. Theimmunogen can be administered alone, or mixed with adjuvant, orexpressed from a vector (VEE replicon vector, vaccinia), or as DNA, oras a fusion protein to induce an immune response. Fusion proteinscomprise the peptide against which an immune response is desired coupledto carrier proteins, such as, for example, beta.-galactosidase,glutathione S-transferase, keyhole limpet hemocyanin (KLH), and bovineserum albumin. In these cases, the peptides serve as haptens with thecarrier proteins. After the animal is boosted, for example, two or moretimes, spleen cells are harvested from the immunized animals andhybridomas generated by fusing sensitized spleen cells with a myelomacell line, such as murine SP2/O myeloma cells (ATCC, Manassas, Va.)using the well-known processes of Kohler and Milstein (Nature 256:495-497 (1975)) and Harlow and Lane (Antibodies: A Laboratory Manual(Cold Spring Harbor Laboratory, New York 1988)).

In a specific embodiment of the invention the antigenic constructaccording to the invention, particularly a vaccine compositioncomprising said antigenic construct in a pharmaceutically acceptableform, is administered in repeated doses, in particular in 1 to 15 doses,more particularly in 2 to 10 doses, even more particularly in 3 to 7doses but especially in 4 to 6 doses, in time intervals of between 1 and10 weeks, particularly in time intervals of between 1 and 6 weeks, moreparticularly in time intervals of between 1 and 4 weeks, and even moreparticularly in time intervals of between 2 and 3 weeks. The immuneresponse is monitored by taking sera samples at a suitable time afterboosting, particularly 3 to 10 days after boosting, more particularly 4to 8 days after boosting and more particularly 5 to 6 days afterboosting and determining the immunogenicity of the antigenic constructusing known methodology, particularly one of the commonly usedimmunoassays such as, for example, an ELISA assay

Immunization with the antigenic construct according to the invention,but particularly with a vaccine composition comprising the antigenicconstruct according to the invention in a pharmaceutically acceptableform leads to a significant immune response in the treated animal.Animals, but especially mice with therapeutic titers are selected for afusion of antibody producing cells, particularly B-lymphocytes with acontinuously growing or immortal cell line, such as a myeloma cell line.The cells are induced to fuse by the addition of polyethylene glycol.Therapeutic titers are those which give a positive result in an ELISAassay in a dilution of between 1:4000 and 1:6000, particularly ofbetween 1:4500 and 1:5500, more particularly of 1:5000.

The resulting hybrid cells are then cloned in the conventional manner,e.g. using limiting dilution, and the resulting clones, which producethe desired monoclonal antibodies, cultured.

The so obtained hybridomas are chemically selected by plating the cellsin a selection medium containing hypoxanthine, aminopterin and thymidine(HAT).

Hybridomas are subsequently screened for the ability to producemonoclonal antibodies against specific amyloid-associated diseases ordisorders. Hybridomas producing antibodies of interest are cloned,expanded and stored frozen for future production. The preferredhybridoma produces a monoclonal antibody having the IgG isotype.

The polyclonal antibody is prepared by immunizing animals, such as miceor rabbits, or any other suitable animal with supramolecular antigenicconstruct compositions of the present invention described herein. Bloodsera is subsequently collected from the animals, and antibodies in thesera screened for binding reactivity against the amyloid protein.

The antibodies according to the invention can be prepared in aphysiologically acceptable formulation and may comprise apharmaceutically acceptable carrier, diluent and/or excipient usingknown techniques. For example, the antibody according to the inventionand as described herein including any functionally equivalent antibodyor functional parts thereof, in particular, the monoclonal antibodyincluding any functionally equivalent antibody or functional partsthereof is combined with a pharmaceutically acceptable carrier, diluentand/or excipient to form a therapeutic composition. Suitablepharmaceutical carriers, diluents and/or excipients are well known inthe art and include, for example, phosphate buffered saline solutions,water, emulsions such as oil/water emulsions, various types of wettingagents, sterile solutions, etc.

Formulation of the pharmaceutical composition according to the inventioncan be accomplished according to standard methodology know to those ofordinary skill in the art.

The compositions of the present invention may be administered to asubject in the form of a solid, liquid or aerosol at a suitable,pharmaceutically effective dose. Examples of solid compositions includepills, creams, and implantable dosage units. Pills may be administeredorally. Therapeutic creams may be administered topically. Implantabledosage units may be administered locally, for example, at a tumor site,or may be implanted for systematic release of the therapeuticcomposition, for example, subcutaneously. Examples of liquidcompositions include formulations adapted for injection intramuscularly,subcutaneously, intravenously, intra-arterially, and formulations fortopical and intraocular administration. Examples of aerosol formulationsinclude inhaler formulations for administration to the lungs.

The compositions may be administered by standard routes ofadministration. In general, the composition may be administered bytopical, oral, rectal, nasal, interdermal, intraperitoneal, orparenteral (for example, intravenous, subcutaneous, or intramuscular)routes. In addition, the composition may be incorporated into sustainedrelease matrices such as biodegradable polymers, the polymers beingimplanted in the vicinity of where delivery is desired, for example, atthe site of a tumor. The method includes administration of a singledose, administration of repeated doses at predetermined time intervals,and sustained administration for a predetermined period of time.

A sustained release matrix, as used herein, is a matrix made ofmaterials, usually polymers which are degradable by enzymatic oracid/base hydrolysis or by dissolution. Once inserted into the body, thematrix is acted upon by enzymes and body fluids. The sustained releasematrix desirably is chosen by biocompatible materials such as liposomes,polylactides (polylactide acid), polyglycolide (polymer of glycolicacid), polylactide co-glycolide (copolymers of lactic acid and glycolicacid), polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid,collagen, chondroitin sulfate, carboxylic acids, fatty acids,phospholipids, polysaccharides, nucleic acids, polyamino acids, aminoacids such phenylalanine, tyrosine, isoleucine, polynucleotides,polyvinyl propylene, polyvinylpyrrolidone and silicone. A preferredbiodegradable matrix is a matrix of one of either polylactide,polyglycolide, or polylactide co-glycolide (co-polymers of lactic acidand glycolic acid).

It is well known to those of ordinary skill in the art that the dosageof the composition will depend on various factors such as, for example,the condition of being treated, the particular composition used, andother clinical factors such as weight, size, sex and general healthcondition of the patient, body surface area, the particular compound orcomposition to be administered, other drugs being administeredconcurrently, and the route of administration.

The composition may be administered in combination with othercompositions comprising an biologically active substance or compound,particularly at least one compound selected from the group consisting ofcompounds against oxidative stress, anti-apoptotic compounds, metalchelators, inhibitors of DNA repair such as pirenzepin and metabolites,3-amino-1-propanesulfonic acid (3APS), 1,3-propanedisulfonate (1,3PDS),secretase activators, β- and γ-secretase inhibitors, tau proteins,neurotransmitter, β-sheet breakers, anti-inflammatory molecules,“atypical antipsychotics” such as, for example clozapine, ziprasidone,risperidone, aripiprazole or olanzapine or cholinesterase inhibitors(ChEIs) such as tacrine, rivastigmine, donepezil, and/or galantamine andother drugs and nutritive supplements such as, for example, vitamin B12,cysteine, a precursor of acetylcholine, lecithin, choline, Ginkgobiloba, acyetyl-L-carnitine, idebenone, propentofylline, or a xanthinederivative, together with an antibody according to the present inventionand, optionally, a pharmaceutically acceptable carrier and/or a diluentand/or an excipient and instructions for the treatment of diseases.

Proteinaceous pharmaceutically active matter may be present in amountsbetween 1 ng and 10 mg per dose. Generally, the regime of administrationshould be in the range of between 0.1 μg and 10 mg of the antibodyaccording to the invention, particularly in a range 1.0 μg to 1.0 mg,and more particularly in a range of between 1.0 μg and 100 μg, with allindividual numbers falling within these ranges also being part of theinvention. If the administration occurs through continuous infusion amore proper dosage may be in the range of between 0.01 μg and 10 mgunits per kilogram of body weight per hour with all individual numbersfalling within these ranges also being part of the invention.

Administration will generally be parenterally, e.g. intravenously.Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions and emulsions. Non-aqueous solventsinclude without being limited to it, propylene glycol, polyethyleneglycol, vegetable oil such as olive oil, and injectable organic esterssuch as ethyl oleate. Aqueous solvents may be chosen from the groupconsisting of water, alcohol/aqueous solutions, emulsions or suspensionsincluding saline and buffered media. Parenteral vehicles include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's, or fixed oils. Intravenous vehicles include fluid andnutrient replenishers, electrolyte replenishers (such as those based onRinger's dextrose) and others. Preservatives may also be present suchas, for example, antimicrobials, anti-oxidants, chelating agents, inertgases, etc.

The pharmaceutical composition may further comprise proteinaceouscarriers such as, for example, serum albumin or immunoglobulin,particularly of human origin. Further biologically active agents may bepresent in the pharmaceutical composition of the invention dependent onits the intended use.

When the binding target is located in the brain, certain embodiments ofthe invention provide for the antibody or active fragment thereof totraverse the blood-brain barrier. Certain neurodegenerative diseases areassociated with an increase in permeability of the blood-brain barrier,such that the antibody or active fragment thereof can be readilyintroduced to the brain. When the blood-brain barrier remains intact,several art-known approaches exist for transporting molecules across it,including, but not limited to, physical methods, lipid-based methods,and receptor and channel-based methods.

Physical methods of transporting the antibody or active fragment thereofacross the blood-brain barrier include, but are not limited to,circumventing the blood-brain barrier entirely, or by creating openingsin the blood-brain barrier. Circumvention methods include, but are notlimited to, direct injection into the brain (see, e.g., Papanastassiouet al., Gene Therapy 9: 398-406 (2002)) and implanting a delivery devicein the brain (see, e.g., Gill et al., Nature Med. 9: 589-595 (2003); andGliadel Wafers™, Guildford Pharmaceutical). Methods of creating openingsin the barrier include, but are not limited to, ultrasound (see, e.g.,U.S. Patent Publication No. 2002/0038086), osmotic pressure (e.g., byadministration of hypertonic mannitol (Neuwelt, E. A., Implication ofthe Blood-Brain Barrier and its Manipulation, Vols 1 & 2, Plenum Press,N.Y. (1989))), permeabilization by, e.g., bradykinin or permeabilizerA-7 (see, e.g., U.S. Pat. Nos. 5,112,596, 5,268,164, 5,506,206, and5,686,416), and transfection of neurons that straddle the blood-brainbarrier with vectors containing genes encoding the antibody orantigen-binding fragment (see, e.g., U.S. Patent Publication No.2003/0083299).

Lipid-based methods of transporting the antibody or active fragmentthereof across the blood-brain barrier include, but are not limited to,encapsulating the antibody or active fragment thereof in liposomes thatare coupled to active fragments thereof that bind to receptors on thevascular endothelium of the blood-brain barrier (see, e.g., U.S. PatentApplication Publication No. 20020025313), and coating the antibody oractive fragment thereof in low-density lipoprotein particles (see, e.g.,U.S. Patent Application Publication No. 20040204354) or apolipoprotein E(see, e.g., U.S. Patent Application Publication No. 20040131692).

Receptor and channel-based methods of transporting the antibody oractive fragment thereof across the blood-brain barrier include, but arenot limited to, using glucocorticoid blockers to increase permeabilityof the blood-brain barrier (see, e.g., U.S. Patent ApplicationPublication Nos. 2002/0065259, 2003/0162695, and 2005/0124533);activating potassium channels (see, e.g., U.S. Patent ApplicationPublication No. 2005/0089473), inhibiting ABC drug transporters (see,e.g., U.S. Patent Application Publication No. 2003/0073713); coatingantibodies with a transferrin and modulating activity of the one or moretransferrin receptors (see, e.g., U.S. Patent Application PublicationNo. 2003/0129186), and cationizing the antibodies (see, e.g., U.S. Pat.No. 5,004,697).

In a further embodiment the present invention provides methods and kitsfor the detection and diagnosis of amyloid-associated diseases orconditions, for diagnosing a predisposition to an amyloid-associateddisease or condition or for monitoring minimal residual disease in apatient or for predicting responsiveness of a patient to a treatmentwith an antibody or a vaccine composition according to the invention andas described herein. These methods include known immunological methodscommonly used for detecting or quantifying substances in biologicalsamples or in an in situ condition.

Diagnosis of an amyloid-associated disease or condition or of apredisposition to an amyloid-associated disease or condition in apatient may be achieved by detecting the immunospecific binding of anantibody of the invention, particularly a monoclonal antibody or anactive fragment thereof to an epitope of the amyloid protein in a sampleor in situ, which includes bringing the sample or a specific body partor body area suspected to contain the amyloid protein into contact withan antibody which binds an epitope of the amyloid protein, allowing theantibody to bind to the amyloid protein to form an immunologicalcomplex, detecting the formation of the immunological complex andcorrelating the presence or absence of the immunological complex withthe presence or absence of amyloid protein in the sample or specificbody part or area, optionally comparing the amount of said immunologicalcomplex to a normal control value, wherein an increase in the amount ofsaid immunological complex compared to a normal control value indicatesthat said patient is suffering from or is at risk of developing anamyloid-associated disease or condition. The amyloid protein may be inthe monomeric, fibril, and/or polymeric form. The antibody or activeportion thereof may be specific for the monomeric, fibril, and/orpolymeric forms of the amyloid protein.

Monitoring minimal residual disease in a patient following treatmentwith an antibody or a vaccine composition according to the invention maybe achieved by detecting the immunospecific binding of an antibody ofthe invention, particularly a monoclonal antibody or an active fragmentthereof to an epitope of the amyloid protein in a sample or in situ,which includes bringing the sample or a specific body part or body areasuspected to contain the amyloid protein into contact with an antibodywhich binds an epitope of the amyloid protein, allowing the antibody tobind to the amyloid protein to form an immunological complex, detectingthe formation of the immunological complex and correlating the presenceor absence of the immunological complex with the presence or absence ofamyloid protein in the sample or specific body part or area, optionallycomparing the amount of said immunological complex to a normal controlvalue, wherein an increase in the amount of said immunological complexcompared to a normal control value indicates that said patient may stillsuffer from a minimal residual disease. The amyloid protein may be inthe monomeric, fibril, and/or polymeric form. The antibody or activeportion thereof may be specific for the monomeric, fibril, and/orpolymeric forms of the amyloid protein.

Predicting responsiveness of a patient to a treatment with a vaccinecomposition according to the invention may be achieved by detecting theimmunospecific binding of a monoclonal antibody or an active fragmentthereof to an epitope of the amyloid protein in a sample or in situ,which includes bringing the sample or a specific body part or body areasuspected to contain the amyloid protein into contact with an antibodywhich binds an epitope of the amyloid protein, allowing the antibody tobind to the amyloid protein to form an immunological complex, detectingthe formation of the immunological complex and correlating the presenceor absence of the immunological complex with the presence or absence ofamyloid protein in the sample or specific body part or area, optionallycomparing the amount of said immunological complex before and afteronset of the treatment, wherein an decrease in the amount of saidimmunological complex indicates that said patient has a high potentialof being responsive to the treatment. The amyloid protein may be in themonomeric, fibril, and/or polymeric form. The antibody or active portionthereof may be specific for the monomeric, fibril, and/or polymericforms of the amyloid protein.

Biological samples that may be used in the diagnosis of anamyloid-associated disease or condition, for diagnosing a predispositionto an amyloid-associated disease or condition or for monitoring minimalresidual disease in a patient or for predicting responsiveness of apatient to a treatment with an antibody or a vaccine compositionaccording to the invention and as described herein are, for example,fluids such as serum, plasma, saliva, gastric secretions, mucus,cerebrospinal fluid, lymphatic fluid and the like or tissue or cellsamples obtained from an organism such as neural, brain, cardiac orvascular tissue. For determining the presence or absence of the amyloidprotein in a sample any immunoassay known to those of ordinary skill inthe art. may be used such as, for example, assays which utilize indirectdetection methods using secondary reagents for detection, ELISA's andimmunoprecipitation and agglutination assays. A detailed description ofthese assays is, for example, given in See Harlow and Lane, Antibodies:A Laboratory Manual (Cold Spring Harbor Laboratory, New York 1988555-612, WO96/13590 to Maertens and Stuyver, Zrein et al. (1998) andWO96/29605.

For in situ diagnosis, the antibody or any active and functional partthereof may be administered to the organism to be diagnosed by methodsknown in the art such as, for example, intravenous, intranasal,intraperitoneal, intracerebral, intraarterial injection such that aspecific binding between an antibody according to the invention with aneptitopic region on the amyloid protein may occur. The antibody/antigencomplex may conveniently be detected through a label attached to theantibody or a functional fragment thereof or any other art-known methodof detection.

The immunoassays used in diagnostic applications or in applications fordiagnosing a predisposition to an amyloid-associated disease orcondition or for monitoring minimal residual disease in a patient or forpredicting responsiveness of a patient to a treatment with an antibodyor a vaccine composition according to the invention and as describedherein. typically rely on labelled antigens, antibodies, or secondaryreagents for detection. These proteins or reagents can be labelled withcompounds generally known to those of ordinary skill in the artincluding enzymes, radioisotopes, and fluorescent, luminescent andchromogenic substances including, but not limited to colored particles,such as colloidal gold and latex beads. Of these, radioactive labellingcan be used for almost all types of assays and with most variations.Enzyme-conjugated labels are particularly useful when radioactivity mustbe avoided or when quick results are needed. Fluorochromes, althoughrequiring expensive equipment for their use, provide a very sensitivemethod of detection. Antibodies useful in these assays includemonoclonal antibodies, polyclonal antibodies, and affinity purifiedpolyclonal antibodies.

Alternatively, the antibody may be labelled indirectly by reaction withlabelled substances that have an affinity for immunoglobulin, such asprotein A or G or second antibodies. The antibody may be conjugated witha second substance and detected with a labelled third substance havingan affinity for the second substance conjugated to the antibody. Forexample, the antibody may be conjugated to biotin and theantibody-biotin conjugate detected using labelled avidin orstreptavidin. Similarly, the antibody may be conjugated to a hapten andthe antibody-hapten conjugate detected using labelled anti-haptenantibody.

Those of ordinary skill in the art will know of these and other suitablelabels which may be employed in accordance with the present invention.The binding of these labels to antibodies or fragments thereof can beaccomplished using standard techniques commonly known to those ofordinary skill in the art. Typical techniques are described by Kennedy,J. H., et al., 1976 (Clin. Chim. Acta 70:1-31), and Schurs, A. H. W. M.,et al. 1977 (Clin. Chim Acta 81:1-40). Coupling techniques mentioned inthe latter are the glutaraldehyde method, the periodate method, thedimaleimide method, and others, all of which are incorporated byreference herein.

Current immunoassays utilize a double antibody method for detecting thepresence of an analyte, wherein. the antibody is labeled indirectly byreactivity with a second antibody that has been labeled with adetectable label. The second antibody is preferably one that binds toantibodies of the animal from which the monoclonal antibody is derived.In other words, if the monoclonal antibody is a mouse antibody, then thelabeled, second antibody is an anti-mouse antibody. For the antibody tobe used in the assay described herein, this label is preferably anantibody-coated bead, particularly a magnetic bead. For the antibody tobe employed in the immunoassay described herein, the label is preferablya detectable molecule such as a radioactive, fluorescent or anelectrochemiluminescent substance.

An alternative double antibody system, often referred to as fast formatsystems because they are adapted to rapid determinations of the presenceof an analyte, may also be employed within the scope of the presentinvention. The system requires high affinity between the antibody andthe analyte. According to one embodiment of the present invention, thepresence of the amyloid protein is determined using a pair ofantibodies, each specific for amyloid protein. One of said pairs ofantibodies is referred to herein as a “detector antibody” and the otherof said pair of antibodies is referred to herein as a “captureantibody”. The monoclonal antibody of the present invention can be usedas either a capture antibody or a detector antibody. The monoclonalantibody of the present invention can also be used as both capture anddetector antibody, together in a single assay. One embodiment of thepresent invention thus uses the double antibody sandwich method fordetecting amyloid protein in a sample of biological fluid. In thismethod, the analyte (amyloid protein) is sandwiched between the detectorantibody and the capture antibody, the capture antibody beingirreversibly immobilized onto a solid support. The detector antibodywould contain a detectable label, in order to identify the presence ofthe antibody-analyte sandwich and thus the presence of the analyte.

Exemplary solid phase substances include, but are not limited to,microtiter plates, test tubes of polystyrene, magnetic, plastic or glassbeads and slides which are well known in the field of radioimmunoassayand enzyme immunoassay. Methods for coupling antibodies to solid phasesare also well known to those of ordinary skill in the art. Morerecently, a number of porous material such as nylon, nitrocellulose,cellulose acetate, glass fibers and other porous polymers have beenemployed as solid supports.

The present invention also relates to a diagnostic kit for detectingamyloid protein in a biological sample comprising a composition asdefined above. Moreover, the present invention relates to the latterdiagnostic kit which, in addition to a composition as defined above,also comprises a detection reagent as defined above. The term“diagnostic kit” refers in general to any diagnostic kit known in theart. More specifically, the latter term refers to a diagnostic kit asdescribed in Zrein et al. (1998).

It is still another object of the present invention to provide novelimmunoprobes and test kits for detection and diagnosis ofamyloid-associated diseases and conditions comprising antibodiesaccording to the present invention. For immunoprobes, the antibodies aredirectly or indirectly attached to a suitable reporter molecule, e.g.,an enzyme or a radionuclide. The test kit includes a container holdingone or more antibodies according to the present invention andinstructions for using the antibodies for the purpose of binding toamyloid protein to form an immunological complex and detecting theformation of the immunological complex such that presence or absence ofthe immunological complex correlates with presence or absence of amyloidprotein.

EXAMPLES Example 1 Methods for Making Supramolecular AntigenicConstructs Synthesis of Pegylated β-Amyloid Peptide Antigen

To enhance the immune response, an anchor/spacer has been applied toreconstitute the peptide in the liposome, e.g. polyethylene glycol(PEG). PEG was covalently attached to the lysine residue bound at bothtermini of the peptide. At the other end of the chain (PEG n=70)phosphatidyl ethanol amine (PEA) was covalently bound to function as theanchoring element in the liposome bilayer. Thus, the liposome stillfunctions as an adjuvant and the peptide being sufficiently far awayfrom the bilayer can be processed alone and thus increases itsimmunogenicity as compared to the palmitoylated antigen.

The supramolecular constructs described herein were uniquely synthesizedusing standard Fmoc/tBu amino acid side-chain protections. Typically,pegylation of peptides results in mixtures of regioisomers. Herein aconvenient method for the site-specific attachment of a PEG-lipidconjugate to both the C- and N-terminus of Aβ is demonstrated usingpartially protected peptides.

For those peptide sequences containing internal Lys or His residues(22-35), an orthogonally protected Lys(ivDde) was added to eachterminus. An additional Gly was added to the C-terminal to facilitatesynthesis. The Fmoc group was removed with 20% piperidine in DMF andN-acetylated using acetic anhydride. Selective cleavage of the ivDdegroups was achieved with 3% hydrazine hydrate in DMF for one hour. The2-chlorotrityl resin was favored over the more widely used Wang resinsince the former proved to be much more resistant to hydrazinolysis.Furthermore, the 2-chlorotrityl resin is extremely acid sensitive andthus, unlike the Wang resin, enables the isolation of protectedpeptides. Indeed, it was necessary to perform the coupling reaction inthe solution phase as coupling of the resin-bound peptide to thepre-activated pegylated lipid reagent DSPE-PEG-SPA did not give rise toany coupling product. Thus selective cleavage from the resin under mildconditions (acetic acid/trifluoroethanol/dichloromethane, 1:1:8, 1 h,rt) gave the internally protected peptides.

Solution-phase couplings were achieved successfully with the peptidesderived from sequence Aβ₂₂₋₃₅ and Aβ₂₉₋₄₀, respectively, to DSPE-PEG-SPAin DMSO and excess base. The reactions were then quenched by theaddition of excess ethanolamine for 2 h and the solution lyophilized.

For the sequence 29-40, no special protection strategy was required.

Purification by HPLC (semi-preparative reverse-phase C₄ column) gavebetween 50-70% purity of the N- and C-terminal PEG-lipid conjugateswhose identities were confirmed by MALDI (matrix assisted laserdesorption ionization). Each sequence showed considerable variation inthe ease of the coupling reaction and conditions were adjustedaccordingly (temperature, number of molar equivalents DSPE-PEG-SPA,time). For the separation of excess DSPE-PEG-SPA from the desiredproduct HPLC purification is applied. Separation of the mono- anddi-coupled products before final side-chain deprotections can beachieved by using cation-exchange chromatography. Subsequent peptideside-chain deprotections and separation of the excess quenchedDSPE-PEG-SPA leads to the isolation of the desired conjugates with anacceptable purity.

This approach to the synthesis of N- and C-terminal lipid-PEG β-amyloidantigens using protected peptides is applicable to a wide variety ofpeptide sequences.

Example 2 Antibodies Elicited by Supramolecular Antigenic Constructs

Manufacturing of mAbs raised against Pegylated Aβ₂₂₋₃₅ and Aβ₂₉₋₄₀Supramolecular Antigenic Constructs:

Liposomal antigens were prepared as described (Nicolau et al., 2002,PNAS, 99, 2332-37). The sequences PEG-Aβ₂₂₋₃₅ and -Aβ₂₉₋₄₀ werereconstituted in a construct consisting of liposomes made of dimyristoylphosphatidyl choline (DMPC), dimyristoyl phosphatidyl ethanolamine(DMPEA), dimyristoyl phosphatidyl glycerol (DMPG) and cholesterol(0.9:0.1:0.1:0.7 molar ratios) containing monophosphoryl lipid A (40mg/mM phospholipids). These antigens were used for the immunization inC57BL/6 mice in 2 week intervals. 10-12 animals were immunized with eachantigen. After 3 to 6 boostings, mice with therapeutic titers (when a1:5,000 dilution of the sera were positive in ELISA) were selected for afusion. Spleen cells are harvested from the immunized animals andhybridomas generated by fusing sensitized spleen cells with a myelomacell line. The fusion of the mice's B-lymphocytes from the spleens wasconducted with cells of myeloma cell line SP2-0. (ATCC, Manassas, Va.)using the well-known processes of Kohler and Milstein (Nature 256:495-497 (1975)) and Harlow and Lane (Antibodies: A Laboratory Manual(Cold Spring Harbor Laboratory, New York 1988))

The cells were induced to fuse by the addition of polyethylene glycol.The resulting hybrid cells were then cloned in the conventional manner,e.g. using limiting dilution. IgG producing hybridoma clones wereselected and tested for their specific binding to the Aβ₁₋₄₂ peptide byELISA and the resulting clones, which produce the desired monoclonalantibodies, cultured.

The so obtained hybridomas were chemically selected by plating the cellsin a selection medium containing hypoxanthine, aminopterin and thymidine(HAT).

Hybridomas were subsequently screened for the ability to producemonoclonal antibodies against specific amyloid-associated diseases ordisorders. Hybridomas producing antibodies of interest were cloned,expanded and stored frozen for future production. The preferredhybridomas produce monoclonal antibodies having the IgG isotype.

Example 3 Specificity Determination for Antibody ACI-11-Ab-9 andACI-12-Ab-11

To analyze the specificity of the antibody ACI-11-Ab-9 and ACI-12-Ab-11,different concentrations of pre-formed Amyloid 1-42, 1-40 and 17-40,1-28 fibrils are blotted onto Hybond ECL Nitrocellulose Membrane(Amersham Biosciences). After blocking with 10% dry milk and 0.7% Tween20, membranes are incubated with primary antibody at 20 μg/ml for 2 h atRT. After washing, membranes are incubated with horse radish peroxidaseconjugated sheep anti-mouse IgG antibody (Amersham Biosciences) for 1 hat RT, washed and incubated with cheminluminescent solution followed bythe exposure of the membrane to X-ray film.

To measure binding of the mAb ACI-11-Ab-9 and ACI-12-Ab-11 to amyloidβ1-42, 1-40 and 17-40, 1-28 fibers are pre-formed for seven days at 37°C. and blotted on the membrane. 20 μg/ml antibody is used to measurebinding capacity and the bound antibody is detected by horse radishperoxidase conjugated sheep anti-mouse IgG antibody for 20 minutes ofexposure.

Example 4 Thioflavin T (Th-T) Fluorescent Assay

To measure both inhibition of aggregation as well as disaggregationproperties of the mAbs the Thioflavin T (Th-T) fluorescent assay wasused which specifically binds to fibrillar Aβ₁₋₄₂ molecules andsubsequently the fluorescent emission intensity correlates with theamount of Aβ₁₋₄₂ filaments present in the solution.

Aβ1-42 lyophilized powder was reconstituted in hexafluoroisopropanol(HFIP) to 1 mM. The peptide solution was sonicated for 15 min at roomtemperature, agitated overnight, and aliquots made into non-siliconizedmicrocentrifuge tubes. The HFIP was then evaporated under a stream ofargon. The resulting peptide film was vacuum dried for 10 min and storedat −80° C. until used.

4.1 Aβ1-42 Aggregation Assay

To assay for the antibody-mediated inhibition of Aβ1-42 aggregation theantibody was pre-diluted in PBS and an assay solution containing thefollowing components was made in a non-siliconized incubation tube: 3.3or 0.33 μM pre-diluted antibody, 10 μM thioflavin T, 33 μM Aβ1-42, and8.2% DMSO. Therefore the final molar ratios of antibody to Aβ1-42 were1:10 and 1:100. Appropriate control solutions were also prepared. Thesolutions were then incubated for 24 hrs at 37° C., and thespectrofluorescence (relative fluorescence units; RFU) read in sixreplicates in black 348-well plates (Perkin-Elmer) on a Perkin-ElmerFluoroCount spectrofluorometer. Inhibition of aggregation ordisaggregation is expressed as mean % inhibition or disaggregation,respectively, according to the following equation

${\% \mspace{11mu} {inhibition}} = {\frac{\begin{matrix}{\left( {{{RFU}\mspace{14mu} {of}\mspace{14mu} {pos}\mspace{14mu} {contrl}} - {{RFU}\mspace{14mu} {of}\mspace{14mu} {neg}\mspace{14mu} {contrl}}} \right) -} \\\left( {{{RFU}\mspace{14mu} {of}\mspace{14mu} {sample}\mspace{14mu} {with}\mspace{14mu} A\; \beta \; 1} - 42 -} \right. \\\left. {{{RFU}\mspace{14mu} {of}\mspace{14mu} {sample}\mspace{14mu} {without}\mspace{14mu} A\; {\beta 1}} - 42} \right)\end{matrix}}{\left( {{{RFU}\mspace{14mu} {of}\mspace{14mu} {pos}\mspace{14mu} {contrl}} - {{RFU}\mspace{14mu} {of}\mspace{14mu} {neg}\mspace{14mu} {contrl}}} \right)} \times 100\; \%}$

Antibody ACI-11-Ab-9 showed a significant inhibition of Aβ1-42aggregation as compared to the control. At an antibody to Aβ1-42 molarratio of 1:100 the inhibition averaged 34% (3 independent experiments),whereas at a 1:10 molar ratio the inhibition was 69% (2 independentexperiments).

Antibody ACI-12-Ab-11 also showed a significant inhibition of Aβ1-42aggregation as compared to the control. At an antibody to Aβ1-42 molarratio of 1:100 the inhibition averaged 30% (2 independent experiments),whereas at a 1:10 molar ratio the inhibition was 66% (2 independentexperiments).

4.2 Aβ1-42 Disaggregation Assay

To measure the disaggregation properties of the mAb the Thioflavin T(ThT) fluorescent assay was used which specifically binds to fibrillarAβ₁₋₄₂ molecules and subsequently the fluorescent emission intensitycorrelates with the amount of Aβ₁₋₄₂ filaments present in the solution.

To assay for antibody-mediated disaggregation of pre-aggregated Aβ1-42,a low-molecular weight Aβ1-42, prepared as described above, was made upas a 110 μM solution in 27% DMSO and 1×PBS. This solution was thenallowed to aggregate at 37° C. for 24 hrs after which the following wereadded: 3.3 or 0.33 μM pre-diluted antibody, and 10 μM thioflavin T. Thisresulted in a molar ratio of 1:10 and 1:100 antibody to Aβ1-42,containing 8.2% DMSO. This solution was then incubated for additional 24hrs at 37° C. The spectrofluorescence was then measured and %disaggregation calculated as described above.

Antibody ACI-11-Ab-9 showed a significant disaggregation ofpre-aggregated Aβ1-42 in the disaggregation assay. At an antibody toAβ1-42 molar ratio of 1:100 the disaggregation averaged 22% (3independent experiments), whereas at a 1:10 molar ratio thedisaggregation was 52% (3 independent experiments).

Antibody ACI-12-Ab-11 also showed a significant disaggregation ofpre-aggregated Aβ1-42 in the disaggregation assay. At an antibody toAβ1-42 molar ratio of 1:100 the disaggregation averaged 18% (2independent experiments), whereas at a 1:10 molar ratio thedisaggregation was 54% (2 independent experiments).

From the above results it is evident that antibodies ACI-11-Ab-9 andACI-12-Ab-11 exhibit bi-functionality in interacting with Aβ₁₋₄₂filaments, in that both antibodies are capable of inhibiting aggregationof Aβ1-42 and disaggregation of preformed Aβ1-42 fibers.

Example 5 ACI-11-Ab-9 and ACI-12-Ab-11 Interactions

The interactions between antibodies ACI-11-Ab-9 and ACI-12-Ab-11 withamyloid peptide Aβ₁₋₄₂ is studied using surface plasmon resonance. Thebinding of the mouse antibody to either monomers or fibers of Aβ₁₋₄₂ isdetermined.

All SPR experiments are carried out on a Biacore X instrument (BiacoreAB). Reagents for immobilization (EDC, NHS and ethanolamine), sensorchips CM5 and SA as well as running and sample buffer HBS-EP arepurchased from Biacore AB. Sodium acetate (10 mM, pH 5.0) is used ascoupling buffer to increase coupling yield. Fibrillar Aβ₁₋₄₂ (BAchem) isprepared by adding PBS buffer to Aβ₁₋₄₂ to a final concentration of 3mg/ml and leaving the vials at 37° C. for 7 days. Fibrillar Aβ₁₋₄₂ iscoupled to a CM5 sensor chip containing a surface-bound carboxymethyldextran matrix. Biotinylated monomeric Aβ₁₋₄₂ (Bachem) is coupled to aSensor chip SA consisting of carboxymethyl dextran matrix withcovalently attached Streptavidin. Typically four or five concentrationsof mAb are assayed by serial dilutions using running buffer. Injectionsare performed starting from the lowest concentration and are passed overboth fc 1 and 2 at a flow rate of 30 μL/min for 3 min. Flow cell 2 isunderivatised and responses are subtracted from fc 1 to correct forinstrument noise and bulk refractive changes. After injection isfinished, the surfaces are washed immediately with running buffer for 5min. To remove remaining bound antibody from the Aβ₁₋₄₂ fibrils, surfaceregeneration is performed by injecting pulses of 10 mM NaOH. Kineticanalysis is performed using algorithms for numerical integration andglobal analysis using BIAevaluation 3.0. The curves obtained forinjections of analyte at different concentrations are overlaid and thebaselines adjusted to zero. For curve fitting, all data are fitsimultaneously to a 1:1 homogeneous complex.

Binding of the mouse ACI-11-Ab-9 and ACI-12-Ab-11 antibodies to amyloidis determined.

Example 6 Binding of ACI-11-Ab-9 and ACI-12-Ab-11 Monoclonal Antibody toAmyloid Fibers

To analyze the molecular binding side of antibodies ACI-11-Ab-9 andACI-12-Ab-11 on pre-formed fibers negatively contrasted transmissionelectronic microscopy (TEM) is performed.

The antibodies ACI-11-Ab-9 and ACI-12-Ab-11 are coupled with 8 nmcolloidal gold according to Slot J W, Geuze H J (1985). For theco-incubation of amyloid 1-42 (Aβ1-42) fibers 6.6504 fibers areincubated for 24 h at RT with the gold-labeled antibody with the molarratio of 1:100. Subsequently 5 μl of sample are incubated on the freshglow-discharged Cu grid (mesh 200) covered with parlodium/C film for 45seconds, washed 3 times with water and 1 times with 2% fresh diluted andfiltered uranyl acetate. Samples are stained in 2% uranyl acetate for15-20 sec. Excess of stain on the grids is sucked and consequentlyair-dried. Three grids of each sample are prepared. The grids areanalyzed in transmission electron microscopy Hitachi 7000.

Example 7 Fractionation by Density-Gradient Ultracentrifugation

The properties of monoclonal antibodies ACI-11-Ab-9 and ACI-12-Ab-11 ininhibiting Aβ₁₋₄₂ fiber polymerization and disaggregating ofAβ₁₋₄₂-fibers are studied by density-gradient ultracentrifugation(Rzepecki et al., 2004) which is based on the principle to distributebetween differently sized resulting peptide fibers after incubation withand without antibodies followed by a SDS-PAGE sedimentation analysis ona preformed gradient (OptiPrep™) Simultaneous analysis of populations ofpreformed Aβ-fibers, disaggregation and inhibition of aggregationproperties of the co-incubated antibodies, and the binding of theantibodies to the fibers are obvious advantages of this methods.

For the inhibition of Aβ₁₋₄₂ aggregation, Aβ₁₋₄₂ monomers are incubatedwith mAb ACI-11-Ab-9 and ACI-12-Ab-11, respectively, at two differentmolar ratios (molar ratio of monomer Aβ₁₋₄₂ thirty- or hundred-foldhigher than mAb) with the Aβ final concentration of 50 μM. After 24 hrsincubation at 37° C., samples are overlayed over a discontinuousgradient of Optiprep™ and tubes are spun at 259 000 g for 3 hrs at 4° C.15 fractions are harvested (140 μL, each), fraction 1 is the least densefraction from the top of the gradient and fraction 15 is the densestfraction from the bottom of the gradient. The pellet is also taken. Thecollected fractions are analyzed by SDS-PAGE with silver staining. Theconcentration Aβ₁₋₄₂ for inhibition assays is five times less than fordisaggregation assays which decrease amyloid aggregation kinetic andensure measurement within the linear phase.

For the disaggregation of preformed Aβ₁₋₄₂ fibrils by co-incubation withmAb ACI-11-Ab-9 and ACI-12-Ab-11 (at two different molar ratios 1:30 and1:100, mAb+Monomer Aβ₁₋₄₂ with the Aβ final concentration of 246 μM),the samples are incubated for 24 hours at 37° C. After 24 hrs samplesare fractioned by ultracentrifugation and separated by SDS-PAGE asdescribed above and before (Rzepecki et al., 2004).

Example 8 Fluorescent Assay to Assess Inhibition of Aβ₁₋₄₂ FilamentAggregation and Disaggregation of Preformed Aβ₁₋₄₂ Filaments byCo-Incubation with mAb ACI-11-Ab-9 and ACI-12-Ab-11, RespectivelyBIS-ANS Fluorescent Assay

To assess the inhibition properties of the mAb the BIS-ANS (LeVine,2002) fluorescent assay is used which specifically detects the monomeror non-fibrillous population of Aβ₁₋₄₂ filaments. Before fluorescentmeasurement, Aβ₁₋₄₂ monomers are pre-incubated with either buffer,served as control, or mAb ACI-11-Ab-9 and ACI-12-Ab-11 (molar ratio1:100, mAb vs. Aβ₁₋₄₂ peptide) for 14 hours at 37° C. Relativefluorescent units are automatically recorded and results are expressedas changes to the control in percentage.

Example 9 NMR and Fluorescence Characterization of the Interaction ofACI-11-Ab-9 and ACI-12-Ab-11 Monoclonal Antibody with ¹³C-labeledβ-Amyloid 1-42 Peptide

To evaluate the potential mechanism by which the mAb solubilizepre-formed fibers or inhibit fiber formation, a head-to-head-experimentbetween Th-T fluorescent assay and solid-state NMR of U-¹³C Tyr10 andVal12-labeled β-amyloid 1-42 peptide is performed. Therefore the aim ofthis investigation is to follow the β-sheet transition by solid stateNMR spectroscopy in the β-amyloid peptide and in the presence of themonoclonal antibody and to directly compare this with disaggregationcapacity measured by Th-T fluorescent assay.

Solid-state NMR spectroscopy not only detects a transition in thesecondary structure, but it also allows to localize the domains of theAβ₁₋₄₂-peptide which dominate the structural transition. Solid-state NMRhas proven its applicability to the problem as it has contributed to thestructure determination of the Aβ₁₋₄₂-fibers (Petkova et al., 2004,Petkova et al., 2002). In particular the correlation of the ¹³C_(α) and¹³C_(β) chemical shift with the secondary structure (Cornilescu et al.,1999, Luca et al., 2001, Iwadate et al, 1999) is a valuable tool to testchanges of the secondary structure within a peptide.

The synthesis of the peptide labeled including a ¹³C pre-labeled valineat position 12 (¹²Val) and a ¹³C pre-labeled tyrosine at position 10(¹⁰Tyr) is performed by an Fmoc synthesis protocol. Identity and purityof the peptide are confirmed my MALDI mass spectroscopy. The labeledβ-amyloid peptide (1-42) is used to generate fibers by incubating thepeptide solution in PBS buffer for 1 week at 37° C. The major problem,the poor solubility of the amyloid β-peptide in PBS buffer, could besolved in the following manner: The pH value of the PBS buffer istemporarily increased by tiny amounts of ammonia to dissolve the amyloidβ-peptide. The original pH value of the PBS buffer is re-obtained byincubating the sample in the presence of a bigger PBS bath using thevolatile character of ammonia.

To measure the effect of the β-sheet breaking antibodies, solution offibers are incubated with the antibody for 24 hours at 37° C. for bothNMR and Th-T assay. For real-time comparison an aliquot of the samesolution is used for Th-T fluorescent assay and the remaining solutionis lyophilized for the NMR measurements.

The disaggregation capacities of ACI-11-Ab-9 and ACI-12-Ab-11 areanalyzed by co-incubation with pre-formed 13C-labeled amyloid β-fibersusing Th-T fluorescent assay.

To investigate the differences between PBS (control) and mAb incubationeach spectrum is deconvoluted using PeakFit(http://www.systat.com/-products/PeakFit). The lines are well matched byemploying a mixed Lorentzian/Gaussian fitting procedure.

Example 10 Functionality of ACI-11-Ab-9 and ACI-12-Ab-11 on AmyloidFibers

12.1 Modification of Conformation of Aβ1-42 Fibers and Initiation ofDisaggregation after Binding of the ACI-11-Ab-9 and ACI-12-Ab-11Antibody

In order to evaluate the mechanism by which the antibodies are capableto disaggregate preformed beta-amyloid (Aβ₁₋₄₂) fibers a head-to-headcomparison of Thioflavin-T (Th-T) fluorescent assay is performedmeasuring disaggregation and solid-state Nuclear Magnetic Resonance(NMR) of U-¹³C Tyrosine 10 and Valine 12-labelled Aβ1-42 peptideanalysing secondary conformation.

Example 11 Epitope Mapping of Monoclonal Antibodies ACI-11-Ab-9 andACI-12-Ab-11

Epitope mapping of the monoclonal antibodies ACI-11-Ab-9 andACI-12-Ab-11 was performed by ELISA using a peptide library comprising atotal of 33 biotinylated peptides covering the complete amino acid (aa)sequence of Aβ1-42 (produced by Mimotopes, Clayton Victoria, Australiaand purchased from ANAWA Trading SA, Wangen Switzerland). The peptidesin the peptide library were composed of 8, 9 or 10-mer aa peptides. Abiotinylated complete Aβ1-42 peptide (human sequence) was used aspositive control (Bachem, Bubendorf, Switzerland). In addition, longerpeptides covering Aβ1-28, Aβ17-40, Aβ1-40 and Aβ1-42 were used to definethe broader region to which these antibodies may bind. These 4 peptideswere also biotinlyated (manufactured by Anaspec and purchased from ANAWATrading SA, Switzerland). Epitope mapping was done according to themanufacturer's (Mimotopes) instructions. Briefly, Streptavidin coatedplates (NUNC, Roskilde, Denmark) were blocked with 0.1% BSA in PBSovernight at 4° C. After washing with PBS-0.05% Tween 20, plates werecoated for 1 hour at RT with the different peptides from the library,diluted in 0.1% BSA, 0.1% Sodium Azide in PBS to a final concentrationof 10 μM. After washing, plates were incubated for 1 hour at RT with thedifferent antibodies, diluted to 10 μg/ml for antibodies ACI-11-Ab-9 andACI-12-Ab-11 in 2% BSA, 0.1% Sodium Azide in PBS. Plates were washedagain and incubated with alkaline phosphatase conjugated goat anti mouseIgG (Jackson Immunresearch West Grove, Pa., USA) for 1 h at RT. Afterfinal washing, plates were incubated with phosphatase substrate (pNPP,Sigma-Aldrich, St Louis, Mo., USA) and read after 3 hours of incubationat 405 nm using an ELISA plate reader.

ACI-11-Ab-9 was shown to bind significantly to Aβ1-42, but was unable tobind to any of the short peptides in the peptide library. It istherefore concluded, that the antibody needs more than 8 aa to bind theepitope and/or that ACI-11-Ab-9 may recognize a conformational epitopethat is only present in the full length A β1-42.

ACI-12-Ab-11 also showed significant binding to Ab1-42, but similarly toACI-11-Ab-9, ACI-12-Ab-11 did not bind to any of the short peptides inthe peptide library.

To determine whether antibodies ACI-11-Ab-9 and ACI-12-Ab-11 mayrecognize other Aβ peptides the binding to Aβ1-28, Aβ17-40, and Aβ1-40and Aβ1-42 was evaluated.

ACI-11-Ab-9 showed considerable binding to Aβ1-28, Aβ1-40 and Aβ1-42,but did not show any binding to Aβ17-40.

Taken together, these results suggest that the epitope of ACI-11-Ab-9lies in region 1-28 of Aβ and that the epitope is either longer than 8aa and/or that the binding to Aβ is dependent on the conformation of Aβ.

ACI-12-Ab-11 showed significant binding to Ab1-40 and Ab1-42 but did notshow any binding to Aβ1-28 or to Aβ17-40. Thus, ACI-12-Ab-11 could onlybind to full-length Aβ1-40 and Aβ1-42 and not to any shorter peptides ofAβ. These results suggest that the epitope recognized by ACI-12-Ab-11 isdependent on the conformation of Aβ as even 24- or 28-mers of Aβ wereinsufficient for binding of the antibody.

Example 12 Influence of Passive Vaccination with ACI-11-Ab-9 andACI-12-Ab-11 on Brain Amyloid Load in Single Transgenic hAPP Mice

To assess the in vivo capacity of the ACI-11-Ab-9 and ACI-12-Ab-11monoclonal antibodies to bind and clear soluble amyloid out of thebrain, 6 month old single hAPP mice, gender and age matched, are usedfor a passive immunization study with different dose. Soluble Amyloidload is analyzed at the end of the study by harvesting the brain of theanimals and by performing an Aβ 1-40 and Aβ 1-42 specific ELISA (TGC,Germany).

8-13 animals per group receive two injections at an interval of one weekof 100, 300 and 1000 μg monoclonal antibody in 200 μl PBS whereasinjection of PBS alone serves as control. One day after the secondinjection animals are sacrificed for biochemical analysis of solubleamyloid fraction. To quantify the amount of human Aβ 1-40 and human Aβ1-42 in the soluble fraction of the brain homogenates and/or incerebrospinal fluid (CSF), commercially availableEnzyme-Linked-Immunosorbent-Assay (ELISA) kits are used (h Amyloid β 40or β 42 ELISA high sensitive, TGC, Switzerland). The ELISA is performedaccording to the manufacturer's protocol. Briefly, standards (a dilutionof synthetic Aβ 1-40 or Aβ1-42) and samples are prepared in a 96-wellpolypropylene plate without protein binding capacity (Greiner, Germany).The standard dilutions with final concentrations of 1000, 500, 250, 125,62.5, 31.3 and 15.6 pg/ml and the samples are prepared in the samplediluent, furnished with the ELISA kit, to a final volume of 60 μl. Sinceamyloid levels increase with the age of the mouse and since the actualevaluation requires that the readings of the samples are within thelinear part of the standard curve, the samples for Aβ40 analysis arediluted 2:3, the samples for Aβ42 analysis are not diluted.

Samples, standards and blanks (50 μl) are added to the anti-Aβ-coatedpolystyrol plate (capture antibody selectively recognizes the C-terminalend of the antigen) in addition with a selective anti-Aβ-antibodyconjugate (biotinylated detection antibody) and incubated overnight at4° C. in order to allow formation of theantibody-Amyloid-antibody-complex. The following day, aStreptavidine-Peroxidase-Conjugate is added, followed 30 minutes laterby the addition of a TMB/peroxide mixture, resulting in the conversionof the substrate into a colored product and the color intensity ismeasured by means of photometry with an ELISA-reader with a 450 nmfilter. Quantification of the Aβ content of the samples is obtained bycomparing absorbance to the standard curve made with synthetic Aβ 1-40or Aβ 1-42. Data are expressed as individual changes to mean controlvalue (in percent to control).

Example 13 Influence of Chronic Passive Administration of ACI-11-Ab-9and ACI-12-Ab-11 on Plaque Load in Double Transgenic hAPP×PS1 Mice

To assess the in vivo capacity of the ACI-11-Ab-9 and ACI-12-Ab-11monoclonal antibodies to bind and reduce amyloid plaques in the brain,3.5 month old double transgenic hAPP×PS1 mice, gender and age matched,are used for a 4 month long chronic passive immunization study. Amyloidplaques are analyzed at the end of the study by histochemistry of thebrain of the animals by binding of Thioflavin S.

15 transgenic animals receive 16 weekly injections of 500 μg monoclonalantibody in PBS. 15 animals are injected with PBS alone, serving ascontrols. All injections are given intra-peritoneally. At sacrifice,mice are anaesthetized and flushed trans-cardially with physiologicalserum at 4° C. to remove blood from the brain vessels. Subsequently, thebrain is removed from the cranium and hindbrain and forebrain areseparated with a cut in the coronal/frontal plane. The forebrain isdivided evenly into left and right hemisphere by using a midlinesagittal cut. One hemisphere is post-fixed overnight in 4%paraformaldehyde for histology. Sagittal vibratome sections (40 μm) arecut for free floating incubations and stored at 4° C. until staining inPBS with 0.1% sodium azide. Five sections at different levels arestained for dense plaques with Thioflavin S. Sections of all animalsused are randomized for staining and blind quantification. Images areacquired with a Leica DMR microscope equipped with a Sony DXC-9100Pcamera and analyzed with a computer using Leica Q-Win software. Lightintensity and condenser settings for the microscope are kept constantthroughout the image acquisition process. All acquired images aresubjected to the same computer subroutines to minimize investigatorbias. Density slice thresholding is applied uniformly throughoutanalysis. The area of the subiculum is selected for automaticquantification of the amyloid load in the Thioflavin S staining

Example 14 Influence of Passive Vaccination with ACI-11-Ab-9 andACI-12-Ab-11 on Memory Capacity in Single Transgenic hAPP Mice

To analyze the in vivo capacity of the ACI-11-Ab-9 and ACI-12-Ab-11antibodies to modify or increase cognitive functionality, 9 month oldsingle hAPP mice, gender and age matched, are used for passiveimmunization study. Non-spatial cognition is measured at the end of theimmunization period assed by new Object Recognition Task (ORT).

12 animals per group receive two intra peritoneal injections of 400 μgmonoclonal antibody in 200 μl PBS whereas injection of PBS alone servesas control. One day after the second injection cognitive capability arestudied in a new Object Recognition Task (ORT)^(12,13). For ORTenrollment mice are placed for 10 minutes into a behavioral arena andfaced to a new unknown object. Exploration time is recorded. Three hourslater the same animals are re-placed into the same arena for a 2^(nd)session but faced with the old, previously explored, and additionallywith a new object. Again, exploration times for both objects arerecorded and resulting cognition index is calculated as the ratio ofexploration time for the new object related to total exploration timeand expressed as proportional changes to the control.

Example 15 Binding of the Mouse Monoclonal Antibody to Proto-Fibrillar(PF) Oligomer Enriched Fractions of Aβ 1-42 Peptide Over Low-MolecularWeight (LMW) Monomers

The binding of mouse anti-amyloid beta monoclonal antibodies to lowmolecular weight (LMW) monomer and high molecular weight proto-fibrillar(PF) preparations of Aβ1-42 peptide may be performed using ELISA.

Size exclusion chromatography (SEC) using 2 SEC columns, Superdex 75 HR10/30 (Range 3-70 kDa) and Superose 6 HR 10/30 (Range 5-5,000 kDa), areused to prepare Aβ1-42 peptide fractions consisting of high-molecularweight (HMW) proto-fibrillar (PF) oligomer enriched and low-molecularweight (LMW) monomer preparations of Aβ1-42 peptide. The resultingeluates are then stained with uranyl acetate and are examined byhigh-resolution transmission electron microscopy (TEM) at 100 kV toverify the structural morphology of the eluted Aβ1-42 fractions.

An ELISA is then performed by coating the Aβ1-42 fractions ontohigh-binding assay plate at 2 μM over night. The coated plate is thenblocked with 1.0% BSA and the ACI-24-Ab-3 (mouse EJ1A9) antibody isadded in a serial dilution starting at 20 μg/ml. A serial dilution of astandard antibody (6E10, Chemicon) is also used. Anti-mouse IgG antibodyconjugated to alkaline phosphatase and 4-nitrophenyl phosphate are usedfor detection of binding. Plates are read at 405 nm. All conditions areassayed in duplicate with coefficient of variation (CV)<0.2.

Example 16 Binding of ACI-12-Ab-11 Monoclonal Antibody (Clone FK2A6A6)to Monomer- and Oligomer-Enriched Fractions of the Aβ 1-42 Peptide

The binding of the anti-Aβ antibody ACI-12-Ab-11 (clone: FK2A6A6) tomonomers and oligomers of the Aβ1-42 peptide was assessed. Before beingused in the study, the antibody was stored at −80° C. The Aβ1-42 peptide(W. M. Keck Facility, Yale University) was stored as lyophilized powderuntil the day of use. All other materials were from Sigma-Aldrich(Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) unless otherwiseindicated.

To prepare monomers and high molecular weight fractions with improvedoligomer-enrichment of Aβ1-42 peptide, an improved methodology was usedemploying size exclusion chromatography (SEC). Two SEC columns, SupelcoTSK G4000PW-XL (range: 10-1500 kDa; Sigma) and Superose 6 HR 10/30(range 5-5,000 kDa; GE Healthcare Bio-Sciences AB Uppsala, Sweden), wereused to prepare Aβ1-42 peptide fractions enriched in LMW monomer andhigh weight oligomer fractions. The resulting SEC eluates were thenstained with uranyl acetate and examined by high-resolution transmissionelectron microscopy (TEM) at 100 kV to verify the structural morphologyof the Aβ1-42 fractions (data not shown). To investigate the binding ofthe antibody to the Aβ1-42 fractions, an ELISA was performed. Aβ1-42fractions were coated onto high-binding assay plates at 2.2 μM in PBSfor 2 hrs. The coated plates were then washed five times with 0.05%Tween-20 in PBS and blocked with 1.0% BSA. Anti-Aβ antibodies, includingthe control antibody (6E10) were added in a serial dilution starting atindicated concentrations. Anti-mouse IgG antibody conjugated to alkalinephosphatase (Jackson ImmunoResearch, Suffolk, England), and4-nitrophenyl phosphate was used for detection of binding. Plates wereread at 405 nm following a 14 hr incubation at room temperature. Theassay was repeated three times.

FIG. 9 shows the mean (±SEM) optical density (O.D.) values obtained fromthe three separate ELISA assays. Antibody ACI-12-Ab-11 demonstratedsuperior binding to the Aβ1-42 fraction enriched in oligomers ascompared to the fraction not enriched in oligomers and consistingprimarily of Aβ1-42 monomers (FIG. 9A). In comparison, the controlantibody 6E10 bound equally well to both Aβ1-42 fractions (FIG. 9B).Tables 4 and 5 show the O.D. values obtained in ELISA assays 1, 2, and3, for antibodies ACI-12-Ab-11 and 6E10, respectively.

These results indicate that the antibody ACI-12-Ab-11 (clone: FK2A6A6)shows superior binding affinity to oligomer-enriched fractions of Aβ1-42than it does to monomeric Aβ1-42.

Example 17 Inhibition of Aβ₄₂-ApoE4 Binding

The binding of ApoE4 to amyloid and the capacity of the monoclonalantibodies according to the invention to inhibit the interaction betweenApoE4 and Aβ₄₂ peptide are assessed.

Human recombinant ApoE4 is diluted to 200 nM with PBS, and stored in 0.5ml aliquots at −80° C. 1 mg of Aβ₄₂-biotin peptide is resuspended in 20μl of DMSO and then in 1980 μl of PBS/0.1% BSA/0.1% sodium azide toobtain a final solution of 0.5 mg/ml. An ELISA assay is used todetermine the binding of rhApoE4 to Aβ₄₂. rhApoE4 (100 nM) is incubatedfor 3 hrs at 37° C. with Aβ₄₂-biotin (1 μM) to allow the binding of theprotein to the peptide. The mixture is applied on a streptavidin-coatedplate previously washed 3 times with PBST (PBS+0.05% Tween 20). After 1h incubation at room temperature (RT) the plate is washed 3 times withPBST and blocked overnight at 4° C. with PBS containing 0.1% BSA. BoundApoE4 is detected with an IgG1 mouse anti-human ApoE antibody used at adilution of 1:3000 in PBS and applied on the plate for 2.5 hrs at RT.The plate is washed 4 times with PBST and then incubated 1 hour at RTwith the detection antibody, an anti-mouse IgG coupled to AlkalinePhosphatase (AP) at a dilution of 1:5000 in PBS. After a final wash with4×PBST, plates are incubated for 5.5 hrs with AP substrate pNPP(Phosphatase substrate, 4-Nitrophenyl phosphate Disodium saltHexahydrate) and read at 405 nm using an ELISA plate reader. FIG. 10summarizes the experiment.

The ELISA assay is developed by making 8 times 2-fold dilutions of a mixof rhApoE4 (150 nM) and Aβ₄₂-biotin (1.5 μM). The following negativecontrols are added: (1) rhApoE4 alone; (2) Aβ₄₂-biotin; and (3)rhApoE4-Aβ₄₂-biotin (protocol without mouse anti-ApoE4). FIG. 11 showsthat a positive signal is obtained only when both rhApoE4 andAβ₄₂-biotin are present and the complete ELISA protocol is followed.

To optimize the concentrations of rhApoE4 and Aβ₄₂-biotin in the assay,dilutions of rhApoE4 (e.g., 150 nM) are tested with a constantconcentration of Aβ₄₂-biotin (e.g., normal: 1.5 μM or excess: 15 μM).FIG. 12 shows that an excess of Aβ₄₂-biotin dilutes the signal of theELISA assay as less Aβ₄₂-biotin complexed to rhApoE4 binds to the plate.Based on this test an optimal concentration of rhApoE4 is selected.

The concentration of Aβ₄₂-biotin in the assay is optimized using aconstant concentration of rhApoE4 of 100 nM. Dilutions of Aβ₄₂-biotin(e.g., with a starting concentration of 1.5 μM (diluted to lowerconcentrations, for example as low as 1500 nM)) are tested in the ELISAset-up. Based on the results shown in FIG. 13, an optimal concentrationof 1 μM of Aβ₄₂-biotin is selected to determine the effect of themonoclonal antibodies on the binding of rhApoE4 to Aβ₄₂-biotin.

The effect of one or more of the antibodies of the invention on thebinding of rhApoE4 to Aβ₄₂-biotin is assessed using the above-describedELISA assay, but further including the antibody of the invention in thebinding mixture prior to plating. For example, two-fold dilutions of theantibody may be used, starting at a concentration of 50 μg/mL. Theinclusion of the antibody may be at the time when ApoE4 and Aβ₄₂-biotinare first combined, or it may be after (e.g., several hours after) theApoE4 and Aβ₄₂-biotin were first combined. In the former instance, theability of the antibody of the invention to prevent or inhibit theinteraction of ApoE4 and Aβ₄₂-biotin is assessed, whereas in the latterinstance, the ability of the antibody of the invention to disrupt apreexisting complex between ApoE4 and Aβ₄₂-biotin is assessed.

Tables Antigens Used to Raise Mouse Monoclonal Antibodies

TABLE 1 Antibodies and antigenic constructs used for raising saidantibodies Antigen/ Mouse mAb Clone Sequence Linker Anchor AdjuvantACI-11-Ab-9 FG1F9E4 Aβ₂₂₋₃₅ PEG DSPE Lipid A ACI-12-Ab-11 FK2A6A6Aβ₂₉₋₄₀ PEG DSPE Lipid A

TABLE 2 Binding of Aβ peptides to ACI-11-Ab-9 and ACI-12-Ab- 11. Resultsare expressed as O.D. after background subtraction. Antibody AntibodyPeptide ACI-11-Ab-9 ACI-12-Ab-11 1-28¹ Mean 0.53 −0.02 SD 0.06 0.00 SEM0.04 0.00 17-40¹  Mean 0.02 −0.02 SD 0.04 0.01 SEM 0.03 0.00 1-40¹ Mean1.02 0.62 SD 0.39 0.18 SEM 0.27 0.13  1-42A¹ Mean 0.78 0.44 SD 0.12 0.15SEM 0.08 0.11   1-42B² Mean 1.54 1.07 SD 0.38 0.20 SEM 0.27 0.14¹Peptide from Anaspec ²Peptide from Bachem

TABLE 3 Binding of ACI-11-Ab-9 and ACI-12-Ab-11 to 33 overlappingpeptides of Aβ 1-42 as analyzed by ELISA. Antibody Antibody PeptideACI-11-Ab-9 ACI-12-Ab-11 1 0.10 0.10 2 0.10 0.10 3 0.12 0.11 4 0.11 0.105 0.11 0.10 6 0.11 0.10 7 0.11 0.10 8 0.11 0.10 9 0.11 0.10 10 0.13 0.1011 0.11 0.11 12 0.24 0.21 13 0.17 0.15 14 0.16 0.12 15 0.14 0.10 16 0.140.14 17 0.13 0.12 18 0.11 0.10 19 0.10 0.10 20 0.10 0.10 21 0.10 0.09 220.10 0.10 23 0.11 0.10 24 0.10 0.10 25 0.11 0.11 26 0.12 0.12 27 0.130.12 28 0.12 0.11 29 0.20 0.11 30 0.11 0.11 31 0.12 0.11 32 0.12 0.11 330.11 0.11 Aβ1-42 0.80 0.69 Aβ1-42 0.81 0.69 Aβ1-42 0.80 0.69

TABLE 4 Binding of ACI-12-Ab-11 (clone: FK2A6A6) to Monomer- andOligomer- Enriched Preparations of the Aβ 1-42 Peptide Antibody Monomers(O.D.)^(a) Oligomers (O.D.)^(a) dilution^(b) Assay 1 Assay 2 Assay 3Mean SEM Assay 1 Assay 2 Assay 3 Mean SEM 1:1 1.60 1.04 1.37 1.34 0.162.24 1.85 2.27 2.12 0.14 1:2 0.85 0.51 0.75 0.70 0.10 1.33 1.04 1.321.23 0.10 1:4 0.53 0.30 0.45 0.43 0.07 0.70 0.64 0.80 0.71 0.05 1:8 0.290.18 0.25 0.24 0.03 0.44 0.42 0.47 0.44 0.02 1:16 0.25 0.12 0.18 0.180.04 0.30 0.25 0.28 0.28 0.01 1:32 0.19 0.10 0.14 0.14 0.03 0.20 0.160.20 0.18 0.01 1:64 0.15 0.09 0.13 0.12 0.02 0.19 0.14 0.16 0.16 0.011:128 0.14 0.09 0.12 0.12 0.02 0.16 0.14 0.15 0.15 0.01 ^(a)O.D.:optical density at 405 nm ^(b)Starting dilution for ACI-12-Ab-11 (clone:FK2A6A6) was 40 μg/ml

TABLE 5 Binding of the 6E10 control antibody to Monomer- andOligomer-Enriched Preparations of the Aβ 1-42 Peptide Antibody Monomers(O.D.)^(a) Oligomers (O.D.)^(a) dilution^(b) Assay 1 Assay 2 Assay 3Mean SEM Assay 1 Assay 2 Assay 3 Mean SEM 1:1 3.67 3.77 4.04 3.83 0.113.36 3.67 3.89 3.64 0.15 1:2 3.30 3.48 4.00 3.59 0.21 3.39 3.55 3.833.59 0.13 1:4 3.00 3.29 3.52 3.27 0.15 3.10 3.37 3.64 3.37 0.16 1:8 2.673.00 2.80 2.82 0.10 2.73 2.99 3.23 2.98 0.15 1:16 1.78 1.94 2.23 1.980.13 1.78 1.92 2.07 1.92 0.08 1:32 1.18 1.34 1.54 1.36 0.10 1.27 1.301.40 1.32 0.04 1:64 0.81 0.94 1.08 0.94 0.08 0.93 0.88 0.95 0.92 0.021:128 0.64 0.75 0.86 0.75 0.06 0.62 0.61 0.66 0.63 0.02 ^(a)O.D.:optical density at 405 nm ^(b)Starting dilution for 6E10 was 0.5 μg/ml

DEPOSITS

The following hybridoma cell lines were deposited with the “DeutscheSammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ) inBraunschweig, Mascheroder Weg 1 B, 38124 Branuschweig, under theprovisions of the Budapest Treaty:

Hybridoma line Antibody designation designation Deposition dateAccession No FG1F9E4 ACI-11-Ab-9 May 25, 2007 DSM ACC2845 FK2A6A6ACI-12-Ab-11 May 25, 2007 DSM ACC2846

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1.-92. (canceled)
 93. A method for treating a disease or disorder whichis caused by or associated with amyloid or amyloid-like proteins,comprising administering to a subject in need thereof: (a) a monoclonalantibody, or an active fragment thereof, which is produced by hybridomacell line FG1F9E4, deposited on May 25, 2007 and given deposit numberDSM ACC2845; (b) a monoclonal antibody, or an active fragment thereof,which is produced by hybridoma cell line FK2A6A6, deposited on May 25,2007 and given deposit number DSM ACC2846; (c) an anti-amyloid betaantibody, or active fragment thereof, wherein i. complementaritydetermining region (CDR)1 of the light chain variable region has theamino acid sequence of SEQ ID NO:11; ii. CDR2 of the light chainvariable region has the amino acid sequence of SEQ ID NO:12; iii. CDR3of the light chain variable region has the amino acid sequence of SEQ IDNO:13; and iv. at least one CDR of the heavy chain variable region hasan amino acid sequence selected from the group consisting of SEQ IDNO:14, SEQ ID NO:15, and SEQ ID NO:16; (d) an anti-amyloid betaantibody, or active fragment thereof, wherein i. CDR1 of the heavy chainvariable region has the amino acid sequence of SEQ ID NO:14; ii. CDR2 ofthe heavy chain variable region has the amino acid sequence of SEQ IDNO:15; and iii. CDR3 of the heavy chain variable region has the aminoacid sequence of SEQ ID NO:16; or (e) an anti-amyloid beta antibody, oractive fragment thereof, wherein i. CDR1 of the light chain variableregion has the amino acid sequence of SEQ ID NO:11; ii. CDR2 of thelight chain variable region has the amino acid sequence of SEQ ID NO:12;iii. CDR3 of the light chain variable region has the amino acid sequenceof SEQ ID NO:13; iv. CDR1 of the heavy chain variable region has theamino acid sequence of SEQ ID NO:14; v. CDR2 of the heavy chain variableregion has the amino acid sequence of SEQ ID NO:15; and vi. CDR3 of theheavy chain variable region has the amino acid sequence of SEQ ID NO:16.94. The method of claim 93, which comprises administering to the subjectthe monoclonal antibody, or an active fragment thereof, which isproduced by hybridoma cell line FG1F9E4, deposited on May 25, 2007 andgiven deposit number DSM ACC2845.
 95. The method of claim 93, whichcomprises administering to the subject the monoclonal antibody, or anactive fragment thereof, which is produced by hybridoma cell lineFK2A6A6, deposited on May 25, 2007 and given deposit number DSM ACC2846.96. The method of claim 93, which comprises administering to the subjectthe anti-amyloid beta antibody, or active fragment thereof, wherein i.CDR1 of the light chain variable region has the amino acid sequence ofSEQ ID NO:11; ii. CDR2 of the light chain variable region has the aminoacid sequence of SEQ ID NO:12; iii. CDR3 of the light chain variableregion has the amino acid sequence of SEQ ID NO:13; and iv. at least oneCDR of the heavy chain variable region has an amino acid sequenceselected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, andSEQ ID NO:16.
 97. The method of claim 93, which comprises administeringto the subject the anti-amyloid beta antibody, or active fragmentthereof, wherein i. CDR1 of the heavy chain variable region has theamino acid sequence of SEQ ID NO:14; ii. CDR2 of the heavy chainvariable region has the amino acid sequence of SEQ ID NO:15; and iii.CDR3 of the heavy chain variable region has the amino acid sequence ofSEQ ID NO:16.
 98. The method of claim 93, which comprises administeringto the subject the anti-amyloid beta antibody, or active fragmentthereof, wherein i. CDR1 of the light chain variable region has theamino acid sequence of SEQ ID NO:11; ii. CDR2 of the light chainvariable region has the amino acid sequence of SEQ ID NO:12; iii. CDR3of the light chain variable region has the amino acid sequence of SEQ IDNO:13; iv. CDR1 of the heavy chain variable region has the amino acidsequence of SEQ ID NO:14; v. CDR2 of the heavy chain variable region hasthe amino acid sequence of SEQ ID NO:15; and vi. CDR3 of the heavy chainvariable region has the amino acid sequence of SEQ ID NO:16.
 99. Themethod of claim 93, further comprising administering to the subject abiologically active substance selected from the group consisting of ananti-apoptotic compound, a metal chelator, an inhibitor of DNA repair,3-amino-1-propanesulfonic acid (3APS), 1,3-propanedisulfonate (1,3PDS),a secretase activator, a β-secretase inhibitor, a γ-secretase inhibitor,a tau protein, a neurotransmitter, a β-sheet breaker, ananti-inflammatory molecule, and a cholinesterase inhibitor.
 100. Themethod of claim 99, wherein the inhibitor of DNA repair is pirenzepin ora metabolite thereof.
 101. The method of claim 99, wherein thecholinesterase inhibitor is tacrine, rivastigmine, donepezil,galantamine or a nutritive supplement.
 102. The method of claim 93,wherein the subject is human.
 103. A method of treating or alleviatingthe effects of a disease or disorder caused by or associated withamyloid or amyloid-like proteins comprising administering to a subjectin need thereof: (a) a monoclonal antibody, or an active fragmentthereof, which is produced by hybridoma cell line FG1F9E4, deposited onMay 25, 2007 and given deposit number DSM ACC2845; (b) a monoclonalantibody, or an active fragment thereof, which is produced by hybridomacell line FK2A6A6, deposited on May 25, 2007 and given deposit numberDSM ACC2846; (c) an anti-amyloid beta antibody, or active fragmentthereof, wherein i. CDR1 of the light chain variable region has theamino acid sequence of SEQ ID NO:11; ii. CDR2 of the light chainvariable region has the amino acid sequence of SEQ ID NO:12; iii. CDR3of the light chain variable region has the amino acid sequence of SEQ IDNO:13; and iv. at least one CDR of the heavy chain variable region hasan amino acid sequence selected from the group consisting of SEQ IDNO:14, SEQ ID NO:15, and SEQ ID NO:16; (d) an anti-amyloid betaantibody, or active fragment thereof, wherein i. CDR1 of the heavy chainvariable region has the amino acid sequence of SEQ ID NO:14; ii. CDR2 ofthe heavy chain variable region has the amino acid sequence of SEQ IDNO:15; and iii. CDR3 of the heavy chain variable region has the aminoacid sequence of SEQ ID NO:16; or (e) an anti-amyloid beta antibody, oractive fragment thereof, wherein i. CDR1 of the light chain variableregion has the amino acid sequence of SEQ ID NO:11; ii. CDR2 of thelight chain variable region has the amino acid sequence of SEQ ID NO:12;iii. CDR3 of the light chain variable region has the amino acid sequenceof SEQ ID NO:13; iv. CDR1 of the heavy chain variable region has theamino acid sequence of SEQ ID NO:14; v. CDR2 of the heavy chain variableregion has the amino acid sequence of SEQ ID NO:15; and vi. CDR3 of theheavy chain variable region has the amino acid sequence of SEQ ID NO:16,such that the effects of the disease or disorder are treated oralleviated.
 104. The method of claim 103, wherein the disease ordisorder is amyloidosis, a neurological disorder, GuamParkinson-Dementia complex, progressive supranuclear palsy, multiplesclerosis, Creutzfeld Jacob disease, Parkinson's disease, HIV-relateddementia, ALS (amyotropic lateral sclerosis), inclusion-body myositis(IBM), Adult Onset Diabetes; senile cardiac amyloidosis; endocrinetumors, or macular degeneration.
 105. The method of claim 104, whereinthe neurological disorder is Alzheimer's Disease.
 106. The method ofclaim 103, wherein the disease or disorder is a condition characterizedby a loss of cognitive memory capacity.
 107. The method of claim 106,wherein the condition characterized by a loss of cognitive memorycapacity is mild cognitive impairment (MCI), Lewy body dementia, Down'ssyndrome, or hereditary cerebral hemorrhage with amyloidosis (Dutchtype).
 108. The method of claim 103, which comprises administering tothe subject the monoclonal antibody, or an active fragment thereof,which is produced by hybridoma cell line FG1F9E4, deposited on May 25,2007 and given deposit number DSM ACC2845.
 109. The method of claim 103,which comprises administering to the subject the monoclonal antibody, oran active fragment thereof, which is produced by hybridoma cell lineFK2A6A6, deposited on May 25, 2007 and given deposit number DSM ACC2846.110. The method of claim 103, which comprises administering to thesubject the anti-amyloid beta antibody, or active fragment thereof,wherein i. CDR1 of the light chain variable region has the amino acidsequence of SEQ ID NO:11; ii. CDR2 of the light chain variable regionhas the amino acid sequence of SEQ ID NO:12; iii. CDR3 of the lightchain variable region has the amino acid sequence of SEQ ID NO:13; andiv. at least one CDR of the heavy chain variable region has an aminoacid sequence selected from the group consisting of SEQ ID NO:14, SEQ IDNO:15, and SEQ ID NO:16.
 111. The method of claim 103, which comprisesadministering to the subject the anti-amyloid beta antibody, or activefragment thereof, wherein i. CDR1 of the heavy chain variable region hasthe amino acid sequence of SEQ ID NO:14; ii. CDR2 of the heavy chainvariable region has the amino acid sequence of SEQ ID NO:15; and iii.CDR3 of the heavy chain variable region has the amino acid sequence ofSEQ ID NO:16.
 112. The method of claim 103, which comprisesadministering to the subject the anti-amyloid beta antibody, or activefragment thereof, wherein i. CDR1 of the light chain variable region hasthe amino acid sequence of SEQ ID NO:11; ii. CDR2 of the light chainvariable region has the amino acid sequence of SEQ ID NO:12; iii. CDR3of the light chain variable region has the amino acid sequence of SEQ IDNO:13; iv. CDR1 of the heavy chain variable region has the amino acidsequence of SEQ ID NO:14; v. CDR2 of the heavy chain variable region hasthe amino acid sequence of SEQ ID NO:15; and vi. CDR3 of the heavy chainvariable region has the amino acid sequence of SEQ ID NO:16.
 113. Themethod of claim 103, further comprising administering to the subject abiologically active substance selected from the group consisting of ananti-apoptotic compound, a metal chelator, an inhibitor of DNA repair,3-amino-1-propanesulfonic acid (3APS), 1,3-propanedisulfonate (1,3PDS),a secretase activator, a β-secretase inhibitor, a γ-secretase inhibitor,a tau protein, a neurotransmitter, a β-sheet breaker, ananti-inflammatory molecule, and a cholinesterase inhibitor.
 114. Themethod of claim 113, wherein the inhibitor of DNA repair is pirenzepinor a metabolite thereof.
 115. The method of claim 113, wherein thecholinesterase inhibitor is tacrine, rivastigmine, donepezil,galantamine or a nutritive supplement.
 116. The method of claim 103,wherein the subject is human.
 117. A method for reducing the plaque loadin the brain of a subject suffering from a disease or conditionassociated with an increased plaque load in the brain comprisingadministering to the subject a therapeutically effective amount of: (a)a monoclonal antibody, or an active fragment thereof, which is producedby hybridoma cell line FG1F9E4, deposited on May 25, 2007 and givendeposit number DSM ACC2845; (b) a monoclonal antibody, or an activefragment thereof, which is produced by hybridoma cell line FK2A6A6,deposited on May 25, 2007 and given deposit number DSM ACC2846; (c) ananti-amyloid beta antibody, or active fragment thereof, wherein i. CDR1of the light chain variable region has the amino acid sequence of SEQ IDNO:11; ii. CDR2 of the light chain variable region has the amino acidsequence of SEQ ID NO:12; iii. CDR3 of the light chain variable regionhas the amino acid sequence of SEQ ID NO:13; and iv. at least one CDR ofthe heavy chain variable region has an amino acid sequence selected fromthe group consisting of SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16;(d) an anti-amyloid beta antibody, or active fragment thereof, whereini. CDR1 of the heavy chain variable region has the amino acid sequenceof SEQ ID NO:14; ii. CDR2 of the heavy chain variable region has theamino acid sequence of SEQ ID NO:15; and iii. CDR3 of the heavy chainvariable region has the amino acid sequence of SEQ ID NO:16; or (e) ananti-amyloid beta antibody, or active fragment thereof, wherein i. CDR1of the light chain variable region has the amino acid sequence of SEQ IDNO:11; ii. CDR2 of the light chain variable region has the amino acidsequence of SEQ ID NO:12; iii. CDR3 of the light chain variable regionhas the amino acid sequence of SEQ ID NO:13; iv. CDR1 of the heavy chainvariable region has the amino acid sequence of SEQ ID NO:14; v. CDR2 ofthe heavy chain variable region has the amino acid sequence of SEQ IDNO:15; and vi. CDR3 of the heavy chain variable region has the aminoacid sequence of SEQ ID NO:16.
 118. The method of claim 117, whichcomprises administering to the subject the monoclonal antibody, or anactive fragment thereof, which is produced by hybridoma cell lineFG1F9E4, deposited on May 25, 2007 and given deposit number DSM ACC2845.119. The method of claim 117, which comprises administering to thesubject the monoclonal antibody, or an active fragment thereof, which isproduced by hybridoma cell line FK2A6A6, deposited on May 25, 2007 andgiven deposit number DSM ACC2846.
 120. The method of claim 117, whichcomprises administering to the subject the anti-amyloid beta antibody,or active fragment thereof, wherein i. CDR1 of the light chain variableregion has the amino acid sequence of SEQ ID NO:11; ii. CDR2 of thelight chain variable region has the amino acid sequence of SEQ ID NO:12;iii. CDR3 of the light chain variable region has the amino acid sequenceof SEQ ID NO:13; and iv. at least one CDR of the heavy chain variableregion has an amino acid sequence selected from the group consisting ofSEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16.
 121. The method of claim117, which comprises administering to the subject the anti-amyloid betaantibody, or active fragment thereof, wherein i. CDR1 of the heavy chainvariable region has the amino acid sequence of SEQ ID NO:14; ii. CDR2 ofthe heavy chain variable region has the amino acid sequence of SEQ IDNO:15; and iii. CDR3 of the heavy chain variable region has the aminoacid sequence of SEQ ID NO:16.
 122. The method of claim 117, whichcomprises administering to the subject the anti-amyloid beta antibody,or active fragment thereof, wherein i. CDR1 of the light chain variableregion has the amino acid sequence of SEQ ID NO:11; ii. CDR2 of thelight chain variable region has the amino acid sequence of SEQ ID NO:12;iii. CDR3 of the light chain variable region has the amino acid sequenceof SEQ ID NO:13; iv. CDR1 of the heavy chain variable region has theamino acid sequence of SEQ ID NO:14; v. CDR2 of the heavy chain variableregion has the amino acid sequence of SEQ ID NO:15; and vi. CDR3 of theheavy chain variable region has the amino acid sequence of SEQ ID NO:16.123. The method of claim 117, further comprising administering to thesubject a biologically active substance selected from the groupconsisting of an anti-apoptotic compound, a metal chelator, an inhibitorof DNA repair, 3-amino-1-propanesulfonic acid (3APS),1,3-propanedisulfonate (1,3PDS), a secretase activator, a β-secretaseinhibitor, a γ-secretase inhibitor, a tau protein, a neurotransmitter, aβ-sheet breaker, an anti-inflammatory molecule, and a cholinesteraseinhibitor.
 124. The method of claim 123, wherein the inhibitor of DNArepair is pirenzepin or a metabolite thereof.
 125. The method of claim123, wherein the cholinesterase inhibitor is tacrine, rivastigmine,donepezil, galantamine or a nutritive supplement.
 126. The method ofclaim 117, wherein the subject is human.
 127. A method for reducing theamount of plaques in the brain of a subject suffering from a disease orcondition associated with an increased amount of plaques in the braincomprising administering to the subject a therapeutically effectiveamount of: (a) a monoclonal antibody, or an active fragment thereof,which is produced by hybridoma cell line FG1F9E4, deposited on May 25,2007 and given deposit number DSM ACC2845; (b) a monoclonal antibody, oran active fragment thereof, which is produced by hybridoma cell lineFK2A6A6, deposited on May 25, 2007 and given deposit number DSM ACC2846;(c) an anti-amyloid beta antibody, or active fragment thereof, whereini. CDR1 of the light chain variable region has the amino acid sequenceof SEQ ID NO:11; ii. CDR2 of the light chain variable region has theamino acid sequence of SEQ ID NO:12; iii. CDR3 of the light chainvariable region has the amino acid sequence of SEQ ID NO:13; and iv. atleast one CDR of the heavy chain variable region has an amino acidsequence selected from the group consisting of SEQ ID NO:14, SEQ IDNO:15, and SEQ ID NO:16; (d) an anti-amyloid beta antibody, or activefragment thereof, wherein i. CDR1 of the heavy chain variable region hasthe amino acid sequence of SEQ ID NO:14; ii. CDR2 of the heavy chainvariable region has the amino acid sequence of SEQ ID NO:15; and iii.CDR3 of the heavy chain variable region has the amino acid sequence ofSEQ ID NO:16; or (e) an anti-amyloid beta antibody, or active fragmentthereof, wherein i. CDR1 of the light chain variable region has theamino acid sequence of SEQ ID NO:11; ii. CDR2 of the light chainvariable region has the amino acid sequence of SEQ ID NO:12; iii. CDR3of the light chain variable region has the amino acid sequence of SEQ IDNO:13; iv. CDR1 of the heavy chain variable region has the amino acidsequence of SEQ ID NO:14; v. CDR2 of the heavy chain variable region hasthe amino acid sequence of SEQ ID NO:15; and vi. CDR3 of the heavy chainvariable region has the amino acid sequence of SEQ ID NO:16.
 128. Themethod of claim 127, which comprises administering to the subject themonoclonal antibody, or an active fragment thereof, which is produced byhybridoma cell line FG1F9E4, deposited on May 25, 2007 and given depositnumber DSM ACC2845.
 129. The method of claim 127, which comprisesadministering to the subject the monoclonal antibody, or an activefragment thereof, which is produced by hybridoma cell line FK2A6A6,deposited on May 25, 2007 and given deposit number DSM ACC2846.
 130. Themethod of claim 127, which comprises administering to the subject theanti-amyloid beta antibody, or active fragment thereof, wherein i. CDR1of the light chain variable region has the amino acid sequence of SEQ IDNO:11; ii. CDR2 of the light chain variable region has the amino acidsequence of SEQ ID NO:12; iii. CDR3 of the light chain variable regionhas the amino acid sequence of SEQ ID NO:13; and iv. at least one CDR ofthe heavy chain variable region has an amino acid sequence selected fromthe group consisting of SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16.131. The method of claim 127, which comprises administering to thesubject the anti-amyloid beta antibody, or active fragment thereof,wherein i. CDR1 of the heavy chain variable region has the amino acidsequence of SEQ ID NO:14; ii. CDR2 of the heavy chain variable regionhas the amino acid sequence of SEQ ID NO:15; and iii. CDR3 of the heavychain variable region has the amino acid sequence of SEQ ID NO:16. 132.The method of claim 127, which comprises administering to the subjectthe anti-amyloid beta antibody, or active fragment thereof, wherein i.CDR1 of the light chain variable region has the amino acid sequence ofSEQ ID NO:11; ii. CDR2 of the light chain variable region has the aminoacid sequence of SEQ ID NO:12; iii. CDR3 of the light chain variableregion has the amino acid sequence of SEQ ID NO:13; iv. CDR1 of theheavy chain variable region has the amino acid sequence of SEQ ID NO:14;v. CDR2 of the heavy chain variable region has the amino acid sequenceof SEQ ID NO:15; and vi. CDR3 of the heavy chain variable region has theamino acid sequence of SEQ ID NO:16.
 133. The method of claim 127,further comprising administering to the subject a biologically activesubstance selected from the group consisting of an anti-apoptoticcompound, a metal chelator, an inhibitor of DNA repair,3-amino-1-propanesulfonic acid (3APS), 1,3-propanedisulfonate (1,3PDS),a secretase activator, a β-secretase inhibitor, a γ-secretase inhibitor,a tau protein, a neurotransmitter, a β-sheet breaker, ananti-inflammatory molecule, and a cholinesterase inhibitor.
 134. Themethod of claim 133, wherein the inhibitor of DNA repair is pirenzepinor a metabolite thereof.
 135. The method of claim 133, wherein thecholinesterase inhibitor is tacrine, rivastigmine, donepezil,galantamine or a nutritive supplement.
 136. The method of claim 127,wherein the subject is human.
 137. A method for decreasing the totalamount of soluble Aβ in the brain of a subject suffering from a diseaseor condition associated with increased concentrations of soluble Aβ inthe brain comprising administering to the subject a therapeuticallyeffective amount of: (a) a monoclonal antibody, or an active fragmentthereof, which is produced by hybridoma cell line FG1F9E4, deposited onMay 25, 2007 and given deposit number DSM ACC2845; (b) a monoclonalantibody, or an active fragment thereof, which is produced by hybridomacell line FK2A6A6, deposited on May 25, 2007 and given deposit numberDSM ACC2846; (c) an anti-amyloid beta antibody, or active fragmentthereof, wherein i. CDR1 of the light chain variable region has theamino acid sequence of SEQ ID NO:11; ii. CDR2 of the light chainvariable region has the amino acid sequence of SEQ ID NO:12; iii. CDR3of the light chain variable region has the amino acid sequence of SEQ IDNO:13; and iv. at least one CDR of the heavy chain variable region hasan amino acid sequence selected from the group consisting of SEQ IDNO:14, SEQ ID NO:15, and SEQ ID NO:16; (d) an anti-amyloid betaantibody, or active fragment thereof, wherein i. CDR1 of the heavy chainvariable region has the amino acid sequence of SEQ ID NO:14; ii. CDR2 ofthe heavy chain variable region has the amino acid sequence of SEQ IDNO:15; and iii. CDR3 of the heavy chain variable region has the aminoacid sequence of SEQ ID NO:16; or (e) an anti-amyloid beta antibody, oractive fragment thereof, wherein i. CDR1 of the light chain variableregion has the amino acid sequence of SEQ ID NO:11; ii. CDR2 of thelight chain variable region has the amino acid sequence of SEQ ID NO:12;iii. CDR3 of the light chain variable region has the amino acid sequenceof SEQ ID NO:13; iv. CDR1 of the heavy chain variable region has theamino acid sequence of SEQ ID NO:14; v. CDR2 of the heavy chain variableregion has the amino acid sequence of SEQ ID NO:15; and vi. CDR3 of theheavy chain variable region has the amino acid sequence of SEQ ID NO:16.138. The method of claim 137, which comprises administering to thesubject the monoclonal antibody, or an active fragment thereof, which isproduced by hybridoma cell line FG1F9E4, deposited on May 25, 2007 andgiven deposit number DSM ACC2845.
 139. The method of claim 137, whichcomprises administering to the subject the monoclonal antibody, or anactive fragment thereof, which is produced by hybridoma cell lineFK2A6A6, deposited on May 25, 2007 and given deposit number DSM ACC2846.140. The method of claim 137, which comprises administering to thesubject the anti-amyloid beta antibody, or active fragment thereof,wherein i. CDR1 of the light chain variable region has the amino acidsequence of SEQ ID NO:11; ii. CDR2 of the light chain variable regionhas the amino acid sequence of SEQ ID NO:12; iii. CDR3 of the lightchain variable region has the amino acid sequence of SEQ ID NO:13; andiv. at least one CDR of the heavy chain variable region has an aminoacid sequence selected from the group consisting of SEQ ID NO:14, SEQ IDNO:15, and SEQ ID NO:16.
 141. The method of claim 137, which comprisesadministering to the subject the anti-amyloid beta antibody, or activefragment thereof, wherein i. CDR1 of the heavy chain variable region hasthe amino acid sequence of SEQ ID NO:14; ii. CDR2 of the heavy chainvariable region has the amino acid sequence of SEQ ID NO:15; and iii.CDR3 of the heavy chain variable region has the amino acid sequence ofSEQ ID NO:16.
 142. The method of claim 137, which comprisesadministering to the subject the anti-amyloid beta antibody, or activefragment thereof, wherein i. CDR1 of the light chain variable region hasthe amino acid sequence of SEQ ID NO:11; ii. CDR2 of the light chainvariable region has the amino acid sequence of SEQ ID NO:12; iii. CDR3of the light chain variable region has the amino acid sequence of SEQ IDNO:13; iv. CDR1 of the heavy chain variable region has the amino acidsequence of SEQ ID NO:14; v. CDR2 of the heavy chain variable region hasthe amino acid sequence of SEQ ID NO:15; and vi. CDR3 of the heavy chainvariable region has the amino acid sequence of SEQ ID NO:16.
 143. Themethod of claim 137, further comprising administering to the subject abiologically active substance selected from the group consisting of ananti-apoptotic compound, a metal chelator, an inhibitor of DNA repair,3-amino-1-propanesulfonic acid (3APS), 1,3-propanedisulfonate (1,3PDS),a secretase activator, a β-secretase inhibitor, a γ-secretase inhibitor,a tau protein, a neurotransmitter, a β-sheet breaker, ananti-inflammatory molecule, and a cholinesterase inhibitor.
 144. Themethod of claim 143, wherein the inhibitor of DNA repair is pirenzepinor a metabolite thereof.
 145. The method of claim 143, wherein thecholinesterase inhibitor is tacrine, rivastigmine, donepezil,galantamine or a nutritive supplement.
 146. The method of claim 137,wherein the subject is human.